Preservation of bioactive materials by spray drying

ABSTRACT

This invention provides methods and compositions to preserve bioactive materials in a matrix of powder particles. Methods provide high-pressure gas spraying and/or near supercritical spraying of formulations followed by drying in a stream of conditioned gas to form stable powder particles containing bioactive materials.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Divisional application claiming benefit of andpriority to a prior U.S. application Ser. No. 10/412,651, “Preservationof Bioactive Materials by Spray Drying, to Vu Truong-Le, et al., filedApr. 10, 2003, which claims benefit of and priority to a prior U.S.Provisional Application No. 60/372,192, “Method of Spray-DryingTherapeutic Agents Using Supercritical Fluids”, by Vu Truong-Le, filedApr. 11, 2002; and to a prior U.S. Provisional Application No.60/447,683, “Preservation of Bioactive Materials by Spray Drying”, by VuTruong-Le, filed Feb. 14, 2003. The full disclosure of the priorapplications are incorporated herein by reference.

FIELD OF THE INVENTION

The present invention is in the field of preservation of biologicmaterials in storage. In particular, the invention relates to, e.g.,preservation of bioactive molecules in matrices of spray dried powderparticles.

BACKGROUND OF THE INVENTION

Biological materials, such as proteins, peptides, nucleic acids,bacteria, cells, antibodies, enzymes, serums, vaccines, liposomes, andviruses, are generally unstable when stored in media or other liquidsolutions. For example, enveloped viruses such as live influenza virusmanufactured from egg allantoid fluid loose one log of potency, definedas Tissue Culture Infectious Dose (TCID50), in less than two to threeweeks when stored under refrigerated temperature, i.e. approximately 4°C. At room temperature conditions (approximately 25° C.) and at warmertemperatures such as 37° C., the virus looses the such potency in amatter of days to hours, respectively. Lyophilization processes, whereaqueous formulas are frozen then dried by sublimation, are commonly usedto stabilize these biological materials. Spray-drying is another processcommonly used to remove water from biological materials for storage.Substitution of protectant molecules, such as carbohydrates, afterremoval of water can increase stability by preventing chemicaldegradation, denaturation, and growth of microbial contaminants.

In lyophilization (freeze-drying), the biological material is commonlymixed as a solution or suspension with protective agents, frozen, anddehydrated by sublimation and secondary drying. The low temperatures offreezing and drying by sublimation can slow the kinetics of degradationreactions but they can also reduce the ability of protective agents topenetrate certain biological materials. Moreover, the low temperaturesand low surface to volume ratios involved in freeze drying can requirelong drying times.

Lyophilization and secondary drying processes can force a protein orcell, for example, to undergo significant chemical and physical changes.Such changes can result in loss of activity of the protein due toconcentration of salts, precipitation/crystallization, shear stress, pHextremes, and residual moisture remaining through the freeze-drying.Freeze-drying can pierce cells with ice crystals and fail to protectinternal compartments.

The formation of powder particles by grinding or lyophilized cakes or byspray drying is of substantial interest and importance to thebiopharmaceutical industry for preservation of biologically activematerials. Not only can such fine particles provide a convenient storageform for biomaterials such as proteins, non-protein biomolecules(including for example, DNA, RNA, lipids, and carbohydrates), but theycan be substantially dehydrated for long-term storage and rewettable foradministration of the biomaterial for its intended use after the storageperiod. Further, such dried fine particles could be produced in acontrolled diameter range and may be administered as a dried aerosolpower, for example, via the intranasal route, wherein the nasal mucosawould provide for rewetting and resolvation of the biomaterial in apatient. Numerous other uses of such fine and microfine particlescontaining a biomaterial would find use in the art of pharmaceutics,biologics, and particularly in the field of live virus vaccines. Thus,it would be advantageous to develop methods of forming fine particlescontaining biologically active materials.

Spray drying is a well known process long used, e.g., in the foodprocessing industry to produce powders. For example, liquid products,such as milk, are sprayed through a nozzle into a stream of hot gassesto produce a powder. The increased surface area exposed in the spraymist, in combination with the high temperatures of the drying gas,provides rapid removal of water from the liquid product. However, suchprocess conditions are often unsuitable for sensitive biologic materialsdue to the shear stress, heat stress, oxidative stress, andconformational changes that can occur with loss of hydration water athigh temperatures. Some of these problems are addressed inpharmaceutical spray drying methods, such as those described in U.S.Pat. No. 5,902,844, Spray Drying of Pharmaceutical FormulationsContaining Amino Acid-Based Materials, to Wilson. In Wilson, peptides insolution with a water soluble polymer are sprayed into a stream ofdrying gas to form a pharmaceutical composition. The presence of thepolymer can protect the peptide from degradation by coating the peptideagainst chemical attacks and by substituting for water of hydration lostduring drying. Certain sensitive peptides and other biologicalmaterials, such as nucleic acids, bacteria, cells, antibodies, enzymes,serums, vaccines, liposomes, and viruses can still be damaged, however,by the heat, shear stress and dehydration of the processes described byWilson, and the like.

Larger and more complex biologics, such as live virus and bacterialvaccines, are well recognized as being among the most unstable products.For example, enveloped viruses such as live influenza virus manufacturedfrom egg allantoid fluid loose one log of potency, defined as TissueCulture Infectious Dose (TCID50), in less than two to three weeks whenstored under refrigerated temperature, i.e. approximately 4° C. At roomtemperature conditions (approximately 25° C.), the virus looses the suchpotency in a matter of days.

A need remains for methods to preserve sensitive biological materials,such as proteins and live viruses in storage, particularly attemperatures above freezing. Methods to prepare dry powder particlesusing processes with quick low temperature drying are desirable to suitthe sensitivities of particular biologic materials. What's more, spraydrying processes that do not require exposure to organic co-solvents canreduce denaturation of sensitive biological structures. Compositionsthat can protect such biologicals in storage would provide benefits inmedicine and scientific research. The present invention provides theseand other features that will become apparent upon review of thefollowing.

SUMMARY OF THE INVENTION

The present invention includes methods, apparatus, and compositions forpreserving bioactive materials in storage. The invention provides, e.g.,spraying of a mixture with a high-pressure gas and/or near supercriticalfluid, and spray drying under conditions that can provide fine drypowder particles with reduced shear or temperature stress on sensitivebioactive materials.

The methods of the invention generally include, e.g., spraying abioactive material in suspension or solution mixed with a high pressuregas or near supercritical gas to provide fine particles under conditionsof lower temperature and lower shear stress than typically experiencedwith ordinary spraying or atomization techniques. The fine droplets inthe spray can be dried, e.g., faster, and at lower temperatures, thanwith ordinary techniques. Methods of the invention can provide, e.g.,the ability to generate ultra fine droplet size, resulting in anincreased droplet surface area to volume ratio for increased evaporationefficiency per given heat input. The method of preparing powderparticles in the invention can comprise, for example, preparing anaqueous suspension or solution of a bioactive material and a polyol,forming a mixture of the solution or suspension with a pressurized gasor near supercritical fluid, spraying ultrafine droplets bydepressurizing the mixture, and drying the droplets into powderparticles by exchanging the spray gases with drying gases, e.g., to byspraying into a drying chamber of the spray dry apparatus.

The bioactive material in suspension or solution, for mixture andspraying in the method can be, e.g., a protein, a peptide, a nucleicacid, bacteria, cells, an antibody, an enzyme, serum, a vaccine,liposomes, a virus, and/or the like. Viruses for bioactive materialsuspensions of the method can include, e.g., influenza virus,parainfluenza virus, respiratory syncytial virus, herpes simplex virus,cytomegalo virus, SARS virus, corona virus family members, humanmetapneumovirus, and Epstein-Bar virus.

The polyol in suspensions or solutions for mixing and spraying in themethod can be, e.g., trehalose, sucrose, sorbose, melezitose, glycerol,fructose, mannose, maltose, lactose, arabinose, xylose, ribose,rhamnose, palactose, glucose, mannitol, xylitol, erythritol, threitol,sorbitol, and raffinose. The suspension or solution can also include,e.g., a polymer, such as starch, starch derivatives, carboxymethylstarch, hydroxyethyl starch (HES), dextran, human serum albumin (HSA),and gelatin, to provide protection to the bioactive material andstructure to the particle. A surfactant, such as, e.g., polyethyleneglycol sorbitan monolaurate, polyoxyethylenesorbitan monooleate, orblock copolymers of polyethylene and polypropylene glycol, can be addedto the suspension or solution to increase the solubility of suspensionor solution constituents, and/or to enhance reconstitution and stabilityof powder particles of the invention. Amino acid additives such asarginine, lysine, glycine, methionine, glutamine, histidine, and thelike, can be useful stabilizers.

The suspensions or solutions of the invention can be mixed with apressurized gas or a gas near supercritical conditions before spraying.High pressure gases called the invention can be, e.g., nitrogen, carbondioxide, oxygen, propane, nitrous oxide, helium, hydrogen, and/or thelike, at pressures ranging from about 100 pounds per square inch (psi)to about 15,000 psi, or about 1000 psi. Near supercritical can mean,e.g., a pressure ranging from about 90 percent and 110 percent of thecritical pressure and/or temperature for the fluid. Where the nearsupercritical fluid is carbon dioxide, a typical pressure can be about1200 psi. The near supercritical fluid in the method can be, e.g.,carbon dioxide, sulfur hexafluoride, chlorofluorocarbons, fluorocarbons,nitrous oxide, xenon, propane, n-pentane, ethanol, nitrogen, water,and/or the like. The temperatures of the high-pressure gas or nearsupercritical fluid before mixture with the suspension or solutiontypically ranges from about 0° C. to about 60° C. A modifier, such asmethanol, ethanol, isopropanol, or acetone can be added to the gas orthe near supercritical fluid to, e.g., affect physical properties of thefluid and/or to influence primary drying of the suspension or solution.

Contact of high pressure or near supercritical gas with the solution orsuspension in the mixture can provide, e.g., certain solvation and/oremulsification effects. For example, a solution of the gas, or liquidphase gas, can be formed in the suspension or solution when the gas issoluble to some significant degree in the suspension or solution.Optionally, the solution or suspension can be, e.g., dissolved to someextent in a liquid phase near supercritical gas. In another aspect, thesuspension or solution can be, e.g., emulsified in the high pressure gasor near critical gas, or can be, e.g., or the high pressure gas or nearcritical gas can be emulsified in the suspension or solution. Control ofsuch solvation or emulsification effects can be provided, e.g., byadjustment of mixture temperature, residence time in the mixing chamber,relative proportions of the solution or suspension and high pressure ornear supercritical gas, flow rates, pressures, solution or suspensionconstituents, the presence of additional solvents, and/or the like.

Forming, a mixture of the high-pressure gas and/or near supercriticalfluid with the suspension or solution can take place, e.g., in a nozzlewith a T-junction, a mixing chamber and/or a capillary restrictor.Forming a mixture can entail flowing the solution or suspension with thepressurized gas or near supercritical fluid through a mixing chamber.The mixing chamber can have passage configurations which, e.g., producevortices or turbulence in the flowing mixture to increase the efficiencyof mixing. Reducing the pressure (expansion) of the mixture to form asuspension of droplets in gas can result from, e.g., passage of themixture through the nozzle and out from a spray orifice outlet of thecapillary restrictor. The capillary restrictor can have, e.g., aninternal diameter less that the mixing chamber; typically less thanabout 1000 um, ranging from about 50 um to about 500 um, or about 100um.

A variety of parameters can be adjusted to modify the average size ofthe droplets. Droplet size can be influenced, e.g., by adjusting thenear supercritical fluid pressure or pressure of the high-pressure gas,adjusting the suspension or solution pressure, adjusting the flow rateof the suspension or solution, choice of the nozzle conduit internaldiameter, adjusting the temperature of the drying gas, adjusting thepressure inside the particle formation vessel, changing theconcentrations of suspension or solution constituents, and/or the like.For example, the suspension or solution can be supplied to the mixingchamber at from about 0.5 ml/min to about 30 ml/min to spray from a 100um internal diameter nozzle orifice; lower rates forming smallerdroplets and faster rates forming larger droplets. In the methods,formation of droplets ranging in mass median diameter from about 1 um toabout 200 um is preferred.

Following spray formation of droplets, primary and secondary dryingconvert the droplets into particles. Primary drying can begin, e.g.,with decompression and expansion of the liquid-gas (solution orsuspension—high pressure or near supercritical gas) mixture to form agaseous suspension of droplets. The gas and evaporated solvents of theexpanded mixture can then be exchanged with a drying gas, such asnitrogen at a temperature ranging from about 5° C. to about 90° C.Drying can include secondary drying, wherein, e.g., residual moisture isfurther reduced after gross primary water removal. Drying can beprovided, e.g., in a cyclonic vortex or by suspension of powderparticles in an updraft of drying gases to form a fluidized bed. Toreduce static buildup and reduce possible particle agglomeration,counter ions can be injected into a chamber of dry or drying particles.Drying gases can be recycled, e.g., by reconditioning in heat exchangersand/or desiccators or condensers. At the end of drying, powder particlescan have, e.g., an average size (MMD) ranging from about 0.5 um to about200 um, or about 1 um to about 150 um, or about 5 um to about 20 um,with a moisture content of less than about 10 weight percent, andbioactive material stability in storage, e.g., for at least about ninemonths at about 25° C. or for at least about 2 years in storage at about4° C. Live viruses, live bacteria, and live cells can retain at leastabout half, or at least about 10 percent of original viability in thepowder particles after processing.

Particles can be transferred in streams of drying gas to chambers fordrying, size separation, coating, collection, and/or the like. Powderparticles can be collected by transferring them to a secondary dryingchamber in a flowing stream of drying gas. The secondary drying chambercan be configured as a cyclonic vortex chamber to allow contact ofparticles with warm chamber surfaces and to extend contact time of theparticles with the drying gas. The particles can be separated by size inthe chamber, e.g., by differential settling. The particles can becoated, e.g., with a polymer to provide a protective coat. The particlescan settle to a collection vessel at the bottom of the chamber toaccumulate before recovery. Total process efficiency can be recovery of50%, 70%, 80%, 90%, or more of the bioactive material mass and/oractivity.

Recovered powder particles can be administered as a particle or as areconstituted solution or suspension. The fine particles produced by themethods of the invention can be reconstituted into a suspension orsolution with a bioactive material concentration greater than theoriginal process suspension or solution. For example, the dried powderparticles can be reconstituted at 2-30 times the concentration of theinitial liquid feed (solution or suspension) without incurringsignificant activity loss or protein denaturation; the reconstitutiontimes for 100 mg/ml solutions can be less than 5 minutes. The powderparticles can be administered, e.g., to a mammal by intramuscular,intraperitoneal, intracerebrospinal, subcutaneous, intra-articular,intrasynovial, intrathecal, oral, topical, inhalation intranasal, and/orpulmonary administration routes.

The methods of the invention can be practiced, e.g., using the apparatusof the invention. The apparatus can have chambers to hold and mix thesuspension or solutions and a pressurized gas or near supercriticalfluid, before spraying the mixture from a nozzle into a particleformation vessel. Particles formed therein can be, e.g., dried in astream of drying gas and/or transferred to secondary drying chambersconfigured to further dry, coat, sieve, size, and/or collect theparticles. In one embodiment of the apparatus, for example, a firstchamber contains the suspension or solution of bioactive material and apolyol, a second chamber contains the high pressure gas and/or nearsupercritical fluid, a mixing chamber is in fluid communication with thefirst chamber through a first conduit and with the second chamberthrough a second conduit, a capillary restrictor provides restrictedfluid communication between the mixing chamber and a particle formationvessel, and a stream of a drying gas flows to dry the fine mist ofdroplets formed when the suspension or solution is mixed with the gasand/or near supercritical fluid in the mixing chamber and is sprayedinto the particle formation vessel. The result can be a preparation ofstable dry fine powder particles containing the bioactive material.

The suspension or solution of in the first chamber can include, e.g., abioactive material, polyol, polymer, and a surfactant. The bioactivematerial can include, e.g., proteins, peptides, nucleic acids, bacteria,cells, antibodies, enzymes, serums, vaccines, liposomes, viruses, and/orthe like. The polyol can be, e.g., trehalose, sucrose, sorbose,melezitose, glycerol, fructose, mannose, maltose, lactose, arabinose,xylose, ribose, rhamnose, palactose, glucose, mannitol, xylitol,erythritol, threitol, sorbitol, raffinose, and/or the like. The polymercan be, e.g., starch, starch derivatives, carboxymethyl starch,hydroxyethyl starch (HES), dextran, human serum albumin (HSA), gelatin,and/or the like. The surfactant can be, e.g., polyethylene glycolsorbitan monolaurate (Tween 20), polyoxyethylenesorbitan monooleate(Tween 80), block copolymers of polyethylene and polypropylene glycol(Pluronic), and/or the like. Amino acids additives such as arginine,lysine, glycine, methionine, glutamine, histidine, and the like can beuseful stabilizers.

The gas and/or near supercritical fluid for mixture with the suspensionor solution in the apparatus can be, e.g., nitrogen, carbon dioxide,oxygen, propane, carbon monoxide, fluorane, nitrous oxide, helium,hydrogen, sulfur hexafluoride, chlorofluorocarbons, fluorocarbons,nitrous oxide, xenon, propane, n-pentane, ethanol, nitrogen, and/orwater.

The suspension or solution, and the high-pressure gas and/or nearsupercritical fluid, can be fed to a nozzle of the apparatus to form amixture that is sprayed into the particle formation vessel of theapparatus. A first flow control means, such as a pump or valve, can beconnected to the first conduit between the first chamber and the mixingchamber to control flow of suspension or solution into the mixingchamber. A second flow control means, such as a pump or valve, can beconnected to the second conduit between the second chamber and themixing chamber to control flow of the gas and/or near supercriticalfluid into the mixing chamber. The inlets into the mixing chamber fromthe first conduit and/or second conduit can be directed at an angle lessthan 90 degrees from an axis of the mixing chamber. A capillaryrestrictor can provide, e.g., back pressure to the flowing mixture andan orifice to spray the mixture from the nozzle. The capillaryrestrictor can have, e.g., an internal diameter less than the mixingchamber; typically, the capillary restrictor can have an internaldiameter ranging from about 50 um to about 1000 um, from about 50 um toabout 500 um, or about 100 um. The nozzle can include, e.g., multiplecapillary constrictors. The nozzle can have intersections for multiplefeed channels to accommodate mixture of more than one gas and/or morethan one liquid feed.

The mist of fine droplets formed as the mixture is sprayed from thenozzle can be dried by a drying gas. The particle formation vessel canact as a secondary drying chamber, or can be in fluid contact with asecondary drying chamber where particles can be transferred and dried bycontact the drying gas and/or chamber surfaces. The dying gas can be,e.g., nitrogen gas controlled for temperature and/or humidity. Thedrying gas (inlet gas) can be, e.g., at a temperature less than a glasstransition temperature of the powder particles.

Residual moisture in the particles can be reduced to stabilizing levelsin a secondary drying chamber. The secondary drying chamber can beconfigured to act as a cyclonic vortex, a fluidized bed of powderparticles, a chamber to spray protective coating material onto powderparticles, a size separation device, and/or a particle collectionvessel. Drying gasses can be recycled through the particle formationvessel and/or secondary drying chamber after removing moisture in acondenser or desiccator. Separation of particles by size in theapparatus can be by, e.g., differential settling, surface impact, orfiltration, to produce powder particles range in average size (MMD) fromabout 1 um to about 150 um, or about 10 um. An ion generator can beincluded in the apparatus to neutralize static charges.

The present invention includes compositions, such as suspensions orsolutions of a bioactive material, a polyol, a polymer additive, anamino acid additive, and/or a surfactant, for mixture with ahigh-pressure gas and/or a near supercritical fluid to form spray driedpowder particles with improved stability. The suspensions or solutionscan include other ingredients, such as buffers, carriers, excipients,and/or stabilizers. In one embodiment, the composition is a suspensionof influenza virus in an aqueous solution of sucrose, HES, and a blockcopolymer of polyethylene and polypropylene glycol (Pluronic).

The suspension or solution formulation can include bioactive material,such as proteins, peptides, nucleic acids, bacteria, cells, antibodies,enzymes, serums, vaccines, liposomes, and viruses. The bioactivematerial can be present in an amount ranging, e.g., from less than about0.00001 weight percent to about 30 weight percent or more of thesuspension or solution. In the case of viral bioactive materials, theviruses can be, e.g., influenza virus, parainfluenza virus, respiratorysyncytial virus, herpes simplex virus, SARS (severe acute respiratorysyndrome) virus, corona virus family members, cytomegalo virus, humanmetapneumovirus, and Epstein-Bar virus. Live viruses can be present inthe suspension or solution in a titer ranging, e.g., from about 10³TCID₅₀ to about 10¹² TCID₅₀/ml, or about 10⁶ TCID₅₀/ml. Viruses can bepresent in the dried particles in an amount, e.g., of about 10²TCID₅₀/g, about 10² TCID₅₀/g, about 10³ TCID₅₀/g, about 10⁴ TCID₅₀/g,about 10⁵ TCID₅₀/g, about 10⁶ TCID₅₀/g, about 10⁷ TCID₅₀/g, about 10⁸TCID₅₀/g, about 10⁹ TCID₅₀/g, about 10¹⁰ TCID⁵⁰/g, or about 10¹¹TCID₅₀/g.

The suspension or solution of the invention can include any of a varietyof non-reducing or reducing polyols, such as, e.g., trehalose, sucrose,sorbose, melezitose, glycerol, fructose, mannose, maltose, lactose,arabinose, xylose, ribose, rhamnose, palactose, glucose, mannitol,xylitol, erythritol, threitol, sorbitol, and raffinose. The polyol canbe present in the suspension or solution, e.g., in an amount rangingfrom about 1 weight percent to about 40 weight percent. In a particularembodiment, the polyol is sucrose present in an amount of about 10weight percent of the suspension or solution.

Polymers can be present in the suspensions or solutions of theinvention. Exemplary polymers are hydrophilic biopolymers, such asstarch, starch derivatives, carboxymethyl starch, hydroxyethyl starch(HES), dextran, human serum albumin (HSA), gelatin, and/or the like.Polymers with a molecular weight ranging from about 1 kDa to about 300kDa are often preferred. Polymers are typically present in suspensionsof the invention in concentrations ranging from about 0.5 weight percentto about 10 weight percent. In one embodiment, the suspension orsolution contains HES at a concentration of about 5 weight percent.

Surfactants can be present in the suspensions or solutions of theinvention, e.g., to enhance the solubility of formulation constituents,aid in spraying fine particles, to stabilize bioactive materials, and/orto improve the reconstitution time of the dried particles. Thesuspensions or solutions of the invention can include nonionicsurfactants, such as alkylphenyl alkoxylates, alcohol alkoxylates, fattyamine alkoxylates, polyoxyethylene glycerol fatty acid esters, castoroil alkoxylates, fatty acid alkoxylates, fatty acid amide alkoxylates,fatty acid polydiethanolamides, lanolin ethoxylates, fatty acidpolyglycol esters, isotridecyl alcohol, fatty acid amides,methylcellulose, fatty acid esters, silicone oils, alkyl polyglycosides,glycerol fatty acid esters, polyethylene glycol, polypropylene glycol,polyethylene glycol/polypropylene glycol block copolymers, polyethyleneglycol alkyl ethers, polypropylene glycol alkyl ethers, polyethyleneglycol/polypropylene glycol ether block copolymers, polyethylene glycolsorbitan monolaurate, and/or polyoxyethylenesorbitan monooleate. Thesuspensions or solutions of the invention can include ionic surfactants,such as alkylarylsulfonates, phenylsulfonates, alkyl sulfates, alkylsulfonates, alkyl ether sulfates, alkyl aryl ether sulfates, alkylpolyglycol ether phosphates, polyaryl phenyl ether phosphates,alkylsulfosuccinates, olefin sulfonates, paraffin sulfonates, petroleumsulfonates, taurides, sarcosides, fatty acids, alkylnaphthalenesulfonicacids, naphthalenesulfonic acids, lignosulfonic acids, condensates ofsulfonated naphthalenes with formaldehyde, condensates of sulfonatednaphthalenes with formaldehyde and phenol, lignin-sulfite waste liquor,alkyl phosphates, quaternary ammonium compounds, amine oxides, andbetaines. Surfactants can be present in the suspensions or solutions inamounts ranging, e.g., from about 0.001 weight percent to about 5 weightpercent, or from about 0.01 weight percent to about 1 weight percent.

The suspension or solution (liquid feed material) of the invention canfurther comprise an amino acid stabilizer additive such as lysine,arginine, glycine, methionine, histidine, ant the like. The suspensionor solution can include a buffer, such as a phosphate salt, a carbonatesalt, a borate salt, an acetate salt, histidine, glycine, a citratesalt, and/or the like, to provide a pH, e.g., from about pH 3 to aboutpH 8. The buffers can be present at a concentration ranging from about 2mM to about 500 mM, as appropriate.

The present invention includes, e.g., articles of manufacture comprisinga container containing dried powder particles prepared by spray drying amixture of high-pressure gas and/or near supercritical gas with asuspension or solution of bioactive material, a polyol, a polymeradditive, and a surfactant.

Definitions

Before describing the present invention in detail, it is to beunderstood that this invention is not limited to particular devices orbiological systems, which can, of course, vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting. As used in this specification and the appended claims, thesingular forms “a”, “an” and “the” include plural referents unless thecontent clearly dictates otherwise. Thus, for example, reference to “asurface” includes a combination of two or more surfaces; reference to“bacteria” includes mixtures of bacteria, and the like.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the invention pertains. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice for testing of the present invention, the preferredmaterials and methods are described herein. In describing and claimingthe present invention, the following terminology will be used inaccordance with the definitions set out below.

“Ambient” temperatures or conditions are those at any given time in agiven environment. Typically, ambient room temperature is approximately22° C., ambient atmospheric pressure, and ambient humidity are readilymeasured and will vary depending on the time of year, weatherconditions, altitude, etc.

“Boiling” refers, e.g., to the rapid phase transition from liquid to gasthat takes place when the temperature of a liquid is above its boilingtemperature. The boiling temperature, as is well known to those skilledin the art, is the temperature at which the vapor pressure of a liquidis equal to the applied pressure.

“Buffer” refers to a buffered solution that resists changes in pH by theaction of its acid-base conjugate components. The pH of the buffer willgenerally be chosen to stabilize the active material of choice, and willbe ascertainable by those in the art. Generally, this will be in therange of physiological pH, although some proteins, can be stable at awider range of pHs, for example acidic pH. Thus, preferred pH ranges arefrom about 1 to about 10, with from about 3 to about 8 beingparticularly preferred; more preferably, from about 6.0 to about 8.0;yet more preferably, from about 7.0 to about 7.4; and most preferably,at about 7.0 to about 7.2. Suitable buffers include a pH 7.2 phosphatebuffer and a pH 7.0 citrate buffer. As will be appreciated by those inthe art, there are a large number of suitable buffers that may be used.Suitable buffers include, but are not limited to, amino acids, potassiumphosphate, sodium phosphate, sodium acetate, histidine-HCl, sodiumcitrate, sodium succinate, ammonium bicarbonate and carbonate.Generally, buffers are used at molarities from about 1 mM to about 2 M,with from about 2 mM to about 1 M being preferred, and from about 10 mMto about 0.5 M being especially preferred, and 25 to 50 mM beingparticularly preferred.

“Degassing” refers to the release of a gas from solution in a liquidwhen the partial pressure of the gas is greater than the appliedpressure. If water is exposed to nitrogen gas at one atmosphere (about760 Torr), and the partial pressure of nitrogen in the waterequilibrates to the gas phase pressure, nitrogen can bubble from thewater if the gas pressure is reduced. This is not boiling, and can oftenoccur at pressures above a pressure that would boil a solvent. Forexample, bottled carbonated soft drinks, with a high partial pressure ofCO₂ gas, bubble rapidly when pressure is reduced by removing the bottlecap.

“Dispersibility” means the degree to which a powder composition can bedispersed (i.e. suspended) in a current of air so that the dispersedparticles can be respired or inhaled into the lungs of a subject. Thus,a powder that is 20% dispersible means that only 20% of the mass of thepowder is suspendable by an inhalation device for inhalation into thelungs.

“Dry” in the context of dried powder compositions refers to residualmoisture content less than about 10%. Dried powder compositions arecommonly dried to residual moistures of 5% or less, or between about 3%and 0.1%. “Dry” in the context of particles for inhalation means thatthe composition has a moisture content such that the particles arereadily dispersible in an inhalation device to form an aerosol.

“Excipients” generally refer to compounds or materials that are added toincrease the stability of the therapeutic agent during the spray freezedry process and afterwards, for long term physical stability andflowability of the powder product. Suitable excipients can be, e.g.,agents that do not thicken or polymerize upon contact with water, arebasically innocuous when inhaled by a patient and do not significantlyinteract with the therapeutic agent in a manner that alters itsbiological activity. Suitable excipients are described below andinclude, but are not limited to, proteins such as human and bovine serumalbumin, gelatin, immunoglobulins, carbohydrates includingmonosaccharides (galactose, D-mannose, sorbose, etc.), disaccharides(lactose, trehalose, sucrose, etc.), cyclodextrins, and polysaccharides(raffinose, maltodextrins, dextrans, etc.); an amino acid such asmonosodium glutamate, glycine, alanine, arginine or histidine, as wellas hydrophobic amino acids (tryptophan, tyrosine, leucine,phenylalanine, etc.); a methylamine such as betaine; an excipient saltsuch as magnesium sulfate; a polyol such as trihydric or higher sugaralcohols, e.g. glycerin, erythritol, glycerol, arabitol, xylitol,sorbitol, and mannitol; propylene glycol; polyethylene glycol;Pluronics; surfactants; and combinations thereof. Excipients can bemultifunctional constituents of solutions or suspensions of invention.

“Glass” or “glassy state” or “glassy matrix,” refers to a liquid thathas a markedly reduced ability to flow, i.e. it is a liquid with a veryhigh viscosity, wherein the viscosity ranges from 10¹⁰ to 10¹⁴pascal-seconds. It can be viewed as a metastable amorphous system inwhich the molecules have vibrational motion but have very slow (almostimmeasurable) rotational and translational components. As a metastablesystem, it is stable for long periods of time when stored well below theglass transition temperature. Because glasses are not in a state ofthermodynamic equilibrium, glasses stored at temperatures at or near theglass transition temperature relax to equilibrium and lose their highviscosity. The resultant rubbery or syrupy, flowing liquid is oftenchemically and structurally destabilized. While a glass can be obtainedby many different routes, it appears to be physically and structurallythe same material by whatever route it was taken. The process used toobtain a glassy matrix for the purposes of this invention is generally asolvent sublimation and/or evaporation technique.

The “glass transition temperature” is represented by the symbol T_(g)and is the temperature at which a composition changes from a glassy orvitreous state to a syrup or rubbery state. Generally T_(g) isdetermined using differential scanning calorimetry (DSC) and isstandardly taken as the temperature at which onset of the change of heatcapacity (Cp) of the composition occurs upon scanning through thetransition. The definition of T_(g) is always arbitrary and there is nopresent international convention. The T_(g) can be defined as the onset,midpoint or endpoint of the transition; for purposes of this inventionwe will use the onset of the changes in Cp when using DSC and DER. Seethe article entitled “Formation of Glasses from Liquids and Biopolymers”by C. A. Angell: Science, 267, 1924-1935 (Mar. 31, 1995) and the articleentitled “Differential Scanning Calorimetry Analysis of GlassTransitions” by Jan P. Wolanczyk: Cryo-Letters, 10, 73-76 (1989). Fordetailed mathematical treatment see “Nature of the Glass Transition andthe Glassy State” by Gibbs and DiMarzio: Journal of Chemical Physics,28, NO. 3, 373-383 (March, 1958). These articles are incorporated hereinby reference.

“Penetration enhancers” are surface active compounds that promotepenetration of a drug through a mucosal membrane or lining and aregenerally used intranasally, intrarectally, and intravaginally.

“Pharmaceutically acceptable” excipients (vehicles, additives) are thosewhich can reasonably be administered to a subject mammal to provide aneffective dose of the active ingredient employed. Preferably, these areexcipients which the Federal Drug Administration (FDA) have to datedesignated as ‘Generally Regarded as Safe’ (GRAS).

“Pharmaceutical composition” refers to preparations which are in such aform as to permit the biological activity of the active ingredients tobe unequivocally effective, and which contain no additional componentswhich are toxic to the subjects to which the composition would beadministered.

A “polyol” is a substance with multiple hydroxyl groups, and includessugars (reducing and nonreducing sugars), sugar alcohols and sugaracids. Preferred polyols herein have a molecular weight which is lessthan about 600 kDa (e.g. in the range from about 120 to about 400 kDa).A “reducing sugar” is a polyol which contains a hemiacetal group thatcan reduce metal ions or react covalently with lysine and other aminogroups in proteins. A “nonreducing sugar” is a sugar which does not havethese properties of a reducing sugar. Examples of reducing sugars arefructose, mannose, maltose, lactose, arabinose, xylose, ribose,rhamnose, galactose and glucose. Nonreducing sugars include sucrose,trehalose, sorbose, melezitose and raffinose. Mannitol, xylitol,erythritol, threitol, sorbitol and glycerol are examples of sugaralcohols. As to sugar acids, these include L-gluconate and metallicsalts thereof.

“Powder” means a composition that consists of finely dispersed solidparticles that are relatively free flowing and capable of being readilydispersed in an inhalation device and subsequently inhaled by a patientso that the particles are suitable for intranasal or pulmonaryadministration via the upper respiratory tract including the nasalmucosa.

“Recommended storage temperature” for a composition is the temperatureat which a powdered drug composition is to be stored to maintain thestability of the drug product over the shelf life of the composition inorder to ensure a consistently delivered dose. This temperature isinitially determined by the manufacturer of the composition and approvedby the governmental agency responsible for approval the composition formarketing (e.g., the Food and Drug Administration in the U.S.). Thistemperature will vary for each approved drug product depending on thetemperature sensitivity of the active drug and other materials in theproduct. The recommended storage temperature will vary from less thanabout 0° to about 40° C., but generally will be ambient temperature,i.e. about 25° C. Usually a drug product will be kept at a temperaturethat is at or below the recommended storage temperature.

A biologically active material “retains its biological activity” in apharmaceutical composition, if the biological activity of thebiologically active material, such as an enzyme, at a given time iswithin about 10% (within the errors of the assay) of the biologicalactivity exhibited at the time the pharmaceutical composition wasprepared as determined in a binding assay, for example. For proteins,such as antibodies, purity by analytical techniques such as sizeexclusion HPLC, FTIR, DSC, CD, ELISA, can be correlated to biologicalactivity. In the case of living viruses, biological activity can beconsidered retained when the viral titer of the composition is withinone log of the initial titer. The assay that is used to determine liveinfluenza virus titer is the Fluorescent Focus Assay (FFA assay). Thetiter from this assay is reported as Fluorescent Focus Unit permilliliter (FFU/ml). One FFU/ml is approximately equal to one TissueCulture Infectious Dose per ml (TCID₅₀/ml). Other “biological activity”assays are elaborated below.

A biologically active material “retains its chemical stability” in apharmaceutical composition, e.g., if the chemical stability at a giventime is such that the biologically active material is considered tostill retain its biological activity as defined above. Alternately,chemical stability can be defined, e.g., as no significant change in thestructure of a biological material as accessed by appropriate analyticaltechniques. Chemical stability can be assessed by detecting andquantifying chemically altered forms of the biologically activematerial. Chemical alteration may involve size modification (e.g.clipping of proteins) which can be evaluated using size exclusionchromatography, SDS-PAGE and/or matrix-assisted laser desorptionionization/time-of-flight mass spectrometry (MALDI/TOF MS), for example.Other types of chemical alteration include charge alteration (e.g.occurring as a result of deamidation) which can be evaluated byion-exchange chromatography, for example.

A biologically active material “retains its physical stability” in apharmaceutical composition if, e.g., it shows no significant increase inaggregation, precipitation and/or denaturation upon visual examinationof color and/or clarity, or as measured by UV light scattering or bysize exclusion chromatography.

A “stable” formulation or composition is one in which the biologicallyactive material therein essentially retains its physical stabilityand/or chemical stability and/or biological activity upon storage.Various analytical techniques for measuring stability are available inthe art and are reviewed, e.g., in Peptide and Protein Drug Delivery,247-301, Vincent Lee Ed., Marcel Dekker, Inc., New York, N.Y., Pubs.(1991) and Jones, A. Adv. Drug Delivery Rev. 10: 29-90 (1993). Stabilitycan be measured at a selected temperature for a selected time period.Trend analysis can be used to estimate an expected shelf life before amaterial has actually been in storage for that time period. For liveinfluenza viruses, stability is defined as the time it takes to loose 1log of FFU/ml or 1 log of TCID50/ml. Preferably, the composition isstable at room temperature (˜25° C.) for at least 3 months, and/orstable at about 2-8° C. for at least 1 year. Furthermore, thecomposition is preferably stable following freezing (to, e.g., −70° C.)and thawing of the composition.

High pressure gas or near supercritical drying, as used herein, refersto removal of a solvent, such as water or organic reagents, from asuspension or solution mixed with a high-pressure gas or a nearsupercritical fluid. The high pressure or supercritical drying caninclude, e.g., mixing of the solution or solvent containing the activeingredient with the pressurized gas or the supercritical fluid to form amixture of liquid and gas, spraying of the suspension or solution bydepressurization, expansion, or degassing of the gas-liquid mixture togenerate fine droplets. Many supercritical fluids such as, for example,supercritical carbon dioxide, may be used in the supercritical dryingprocess.

“Near supercritical fluid” refers to a fluid held at, or within about10%, of a critical point pressure and/or temperature (in degree Kelvin).A critical point is a combination of temperature and pressure wherein asubstance can no longer exist as a liquid if the temperature (criticaltemperature) is increased or the pressure (critical pressure) islowered. The critical temperature is the temperature above which a gascannot be liquefied; the temperature above which a substance cannotexhibit distinct gas and liquid phases for a given pressure. Thecritical pressure is the pressure required to liquefy a gas (vapor) at acritical temperature. For example, the critical pressure and temperatureof carbon dioxide are 74 atmospheres and 31 degrees Centigrade,respectively. Carbon dioxide held at a pressure and temperature aboveits critical point is in a supercritical condition or state. Criticalpressures and temperatures for other substances are provided below:

Fluid Pc (bar) Tc (° C.) Carbon dioxide 74 31 Nitrous oxide 72 36 Sulfurhexafluoride 37 45 Xenon 58 16 Ethylene 51 10 Chlorotrifluoromethane 3929 Ethane 48 32 Trifluoromethane 47 26

In a pharmacological sense, a “therapeutically effective amount” of abiologically active material refers to an amount effective in theprevention or treatment of a disorder wherein a “disorder” is anycondition that would benefit from treatment with the biologically activematerial. This includes chronic and acute disorders or diseasesincluding those pathological conditions which predispose the mammal tothe disorder in question.

“Treatment” refers to both therapeutic treatment and prophylactic orpreventative measures. Those in need of treatment include those alreadywith the disorder as well as those in which the disorder is to beprevented.

“Unit dosage” refers to a receptacle containing a therapeuticallyeffective amount of a composition of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B show droplet sizes from a 100 micron fused silica nozzlewhen sprayed using near supercritical CO2 as a function of distance fromthe nozzle tip.

FIG. 2 shows the droplet sizes from a 100 microns fused silica nozzlewhen sprayed using pressurized nitrogen gas as a function of distancefrom the nozzle tip.

FIG. 3 shows histograms representing effects of spray pressure onparticle size.

FIG. 4 shows the dry powder particle size distribution of a spray driedformulation containing B/Harbin live virus vaccine.

FIG. 5 shows the glass transition temperature of AVO47 formulation usingdifferential scanning calorimeter (DSC).

FIG. 6 shows the morphology of an exemplary spray dried powder.

FIG. 7 shows the x-ray diffraction data of spray dried powderformulation AVO47. The diffraction pattern showed glassy amorphousnature of the AVO47 formulation

FIG. 8 shows the long term stability of live B/Harbin influenza virusspray dried in formulation AVO47a.

FIG. 9 is a diagrammatic drawing of an exemplary supercritical CO2 spraydrying system.

DETAILED DESCRIPTION

The methods, apparatus, and compositions of the present invention canprovide high initial purity and extended storage of bioactive materialsin a matrix of dry powder particles. The method provides, e.g., quickdrying of droplets into particles without high heat by, e.g., mixing aformulation of a bioactive material with a high-pressure gas and/or nearsupercritical gas in a mixing chamber before spraying from a nozzle toproduce a fine mist. Solvents can evaporate rapidly from the mistdroplets leaving dry particles that can further dehydrated in asecondary drying chamber. The formulations of the invention include,e.g., suspensions or solutions of the bioactive material with polyols,polymers, amino acids, and/or surfactants, that can dry into a stablepreservative matrix.

Methods of Preparing Powder Particles

Methods of the invention include, e.g., mixture of a bioactive materialsuspension or solution with a near supercritical fluid and/or highpressure gas, expansion of the mixture to form a fine mist (gaseoussuspension) of droplets, and drying of the droplets to powder particlesin a particle formation vessel and/or secondary drying chamber.Expansion of the suspension or solution from the mixture with the nearsupercritical gas can produce very fine droplets under conditions of lowshear stress, and relatively low temperature. The rapid removal of waterduring the expansion, and the fine particle size, allow relatively milddrying conditions in the particle formation vessel and/or secondarydrying chamber. Low shear spraying, low temperature primary drying,and/or moderate secondary drying conditions can reduce processdegradation of bioactive materials in the powder particles and increasestability of the particles in storage.

Methods of preparing powder particles in the invention include, e.g.,preparation of a solution or suspension, mixture with a high pressuregas and/or a near supercritical fluid, spraying into a particleformation chamber for primary drying, secondary drying of the particles,and recovery of dried stable powder particles. The aqueous suspension orsolution can contain, e.g., a bioactive material, a polyol, a polymer,an amino acid, and a surfactant. The near supercritical fluid can be,e.g., carbon dioxide. The mixture can be formed, e.g., in a mixingchamber adjacent to a capillary restrictor spray nozzle outlet. Theexpansion of gas during spraying can disrupt the suspension or solutioninto fine droplets that dry rapidly. Secondary drying can be by, e.g.,suspension of particles in a vortex or fluidized bed oftemperature/humidity controlled gas. The powder particle product can berecovered, e.g., by settling after sizing.

Preparing a Suspension or Solution

Suspensions or solutions (liquid feed materials) of the invention caninclude, e.g., a bioactive material formulated with a polyol, polymer,surfactant, amino acid, and/or buffer, in an aqueous solution. Theingredients can be combined in a sequence using techniques appropriateto the constituents, as is appreciated by those skilled in the art. Forexample, a bioactive material, such as a virus or bacterium, can be,e.g., concentrated and separated from growth media by centrifugation orfiltration before mixture with a polyol solution to form a suspension.Antibodies can be purified and concentrated, e.g., by affinitychromatography before dissolving into a solution with other formulationingredients. Liquid suspensions or solutions for spraying can beprepared by mixing the bioactive material, polyols, and otherexcipients, in an aqueous solution. Some bioactive materials, such as,e.g., peptides and antibodies, dissolve readily into an aqueoussolution. Other bioactive materials, such as, e.g., bacteria andliposomes can be particles that exist as a suspension. Whether thebioactive material provides a solution or suspension, it is often thesuspension or solution can also include, e.g., a polymer, such asstarch, starch derivatives, carboxymethyl starch, hydroxyethyl starch(HES), dextran, human serum albumin (HSA), and gelatin, to provideprotection to the bioactive material and structurally, then gentlyblended with the bioactive material after cooling.

The bioactive materials of the invention can be, e.g., industrialreagents, analytical reagents, vaccines, pharmaceuticals, therapeutics,and the like. Bioactive materials of the invention include, e.g.,proteins, peptides, nucleic acids, bacteria, cells, antibodies, enzymes,serums, vaccines, liposomes, viruses, and/or the like. The bioactivematerial can be, e.g., living cells and/or viable viruses. The bioactivematerial can be, e.g., nonliving cells or liposomes useful as vaccinesor as delivery vehicles for therapeutic agents. Viral bioactivematerials of the invention can be, e.g., live viruses such as, influenzavirus, parainfluenza virus, respiratory syncytial virus, herpes simplexvirus, SARS virus, corona virus family members, cytomegalovirus, humanmetapneumovirus, Epstein-Barr virus, and/or the like. Preparation stepsfor solution or suspension liquid formulations of these materials canvary depending on the unique sensitivities of each material.

The concentration of bioactive materials in the suspension or solutioncan vary widely, depending, e.g., on the specific activity,concentration of excipients, route of administration, and/or intendeduse of the material. Where the bioactive material is a peptide vaccine,live virus or bacteria, for example, the required concentration ofmaterial can be quite low. Where the bioactive material is, e.g., anantibody for therapeutic administration by inhalation, or a liposome fortopical administration, the required concentration can be higher. Ingeneral, bioactive materials can be present in the solutions orsuspensions of the invention at a concentration, e.g., between less thanabout 1 pg/ml to about 150 mg/ml, from about 5 mg/ml to about 80 mg/ml,or about 50 mg/ml, as appropriate.

The suspensions or solutions of bioactive materials can include, e.g.,any of a variety of polyols. In the methods of the invention, polyolscan provide, e.g., a viscosity enhancing agent to reduce the effects ofshear stress during spraying. The polyols can provide protectivebarriers and chemistries to the dry powder particles of the invention.For example, the polyol, such as sucrose, can physically surround andprotect the bioactive material from exposure to damaging light, oxygen,moisture, and/or the like. The polyols can, e.g., replace water ofhydration lost during drying, to prevent denaturation of biomolecules ofthe material. Although the invention is not limited to any particularpolyols, the suspensions or solutions, and powder particle compositions,can include, e.g., sucrose, trehalose, sorbose, melezitose, sorbitol,stachyose, raffinose, fructose, mannose, maltose, lactose, arabinose,xylose, ribose, rhamnose, galactose, glucose, mannitol, xylitol,erythritol, threitol, sorbitol, glycerol, L-gluconate, and/or the like.Where it is desired that the formulation be freeze-thaw stable, thepolyol is preferably one which does not crystallize at freezingtemperatures (e.g. −20° C.) such that it destabilizes the biologicallyactive material in the formulation. The amount of polyol used in theformulation will vary depending on the nature of the biologically activeagent, other excipients, and intended use. However, the suspensions orsolutions generally include a nonreducing sugar in a concentrationbetween about 1% and 40%; more preferably, between about 1 and 20%. In aparticularly preferred embodiment, the suspension or solution comprisesabout 10% sucrose.

Polymers can be included in the suspensions or solutions of the method,e.g., to provide protective and structural benefits. As with polyols,polymers can provide, e.g., physical and chemical protection to thebioactive materials. The linear or branching strands of polymers canprovide, e.g., increased structural strength to the particlecompositions of the invention. Polymers can be applied as a protectiveand/or time release coat to the outside or powder particles of theinvention. Many polymers are, e.g., highly soluble in water, so they donot significantly hinder reconstitution of powder particles. Manypolymers such as polyvinyl pyrrolidone, polyethylene glycol, poly aminoacids, such poly L-lysines, can significantly enhance reconstitutionrates in aqueous solutions. Polymer protective agents, in the methods ofthe invention can include, e.g., starch and starch derivatives, such asoxidized starch, carboxymethyl starch and hydroxyethyl starch. (HES),hydrolyzed gelatin, unhydrolyzed gelatin, ovalbumin, collagen,chondroitin sulfate, a sialated polysaccharide, actin, myosin,microtubules, dynein, kinetin, human serum albumin, and/or the like.Preferably, HES is used with a molecular weight of between about 100,000and 300,000; and more preferably, about 200,000. Generally, theconcentration of HES will be from about 0.5 to about 10%; morepreferably, between about 1 and 5%. A preferred formulation comprisesabout 5% HES.

The suspension or solution of the invention can include, e.g., asurfactant compatible with the particular bioactive material involved. Asurfactant can enhance solubility of other formulation components toavoid aggregation or precipitation at higher concentrations. Surfaceactive agents can, e.g., lower the surface tension of the suspension orsolution so that bioactive materials are not denatured at gas-liquidinterfaces, and/or so that finer droplets can be formed during spraying.The suspensions or solutions according to the invention comprise betweenabout 0.001 and 5%; and preferably, between about 0.05 and 1%, or about0.2%, of a nonionic surfactant, an ionic surfactant, or a combinationthereof.

Buffers can be added to the formulations of the method, e.g., to providea suitable stable pH to the formulations of the method and compositionsof the invention. Typical buffers of the invention include, e.g., aminoacids, potassium phosphate, sodium phosphate, sodium acetate, sodiumcitrate, histidine, glycine, sodium succinate, ammonium bicarbonate,and/or a carbonate. The buffers can be adjusted to the appropriate acidand salt forms to provide, e.g., pH stability in the range from about pH3 to about pH 10, from about pH 4 to about pH 8. A pH near neutral, suchas, e.g., pH 7.2, is preferred for many compositions.

Other excipients can be included in the formulation. For example, aminoacids, such as arginine and methionine can be constituents of theformulation and compositions. The amino acids can, e.g., act aszwitterions that block charged groups on processing surfaces and storagecontainers preventing nonspecific binding of bioactive materials. Theamino acids can increase the stability of compositions by, e.g.,scavenging oxidation agents, scavenging deamidation agents, andstabilizing the conformations of proteins. In another example, glycerolcan be included in the formulations of the invention, e.g., to act as apolyol and/or plasticizer in the powder particle compositions. EDTA canbe included in the composition, e.g., to reduce aggregation offormulation constituents and/or to scavenge metal ions that can initiatedestructive free radical chemistries.

Mixing and Spraying

The suspension or solution of the invention is, e.g., mixed in a chamberwith a high-pressure gas or a near supercritical fluid before sprayingthrough a capillary restrictor nozzle outlet to form a fine mist ofdroplets. Without being bound to a particular theory, the combination ofa high pressure gas or a near supercritical fluid with the suspension orsolution can provide an emulsion mixture of droplets saturated and/orsurrounded with fluid under pressure. As the mixture is released fromthe spray nozzle, the pressure drops rapidly allowing an explosiveexpansion, and/or effervescence (degassing), that disrupts the dropletsinto a fine mist (gaseous suspension of droplets). Such a mist can be,e.g., finer than would result with spraying at a lower pressure (e.g.less than 100 psi) or spraying without a near supercritical fluid. Thedroplets can experience, e.g., cool temperatures during any phasetransition or adiabatic expansion associated with the decompression ofthe mixture. Shear stress can less than with hydraulic spraying (i.e.,spraying liquid without gas) at a pressure high enough to provide thesame fine droplets.

The suspensions or solutions are combined with a near supercriticalfluid and/or high-pressure gas, e.g., in a mixing chamber beforespraying to expand in a particle formation chamber. The suspension orsolution can be held in a container (first chamber) and supplied througha conduit to the mixing chamber. The suspension or solution can beforced into the mixing chamber, e.g., by pressurization of the containeror by pumping through high pressure pump. The high-pressure gas and/ornear supercritical fluid can be supplied to the mixing chamber, e.g.,through a conduit from a pressurized vessel (second chamber). The mixingchamber can be, e.g., an expanded conduit within the nozzle structureconfigured to produce vortices or turbulence in the flowing mixture.Depending, for example, on the gas or fluid, and the suspension orsolution constituents, the bioactive material can exist as a particle,emulsion, precipitate, and/or solute in the mixture.

The spray nozzle of the invention can be adapted to provide the desiredfine mist of droplets. The nozzle can have, e.g., a conduit feeding themixture to a capillary restrictor spray orifice that has an internaldiameter of between about 50 um and about 1000 um, or about 100 um. In apreferred embodiment, the mixture comprises an emulsion of thesuspension or solution in the pressurized gas or near supercriticalfluid, such that when the pressure is rapidly reduced, the fluid rapidlytransitions to gas, dispersing the emulsion droplets. The pressurerelease can be, e.g., rapid enough that the gas formation is explosive,causing the formation of fine droplets comprising the bioactivematerial. More specifically, it has been found that supercritical CO₂assisted spraying results in the generation of ultra fine spraydroplets. The droplet size has been found to vary with distance from thenozzle, as shown in FIGS. 1A and 1B. Without being bound to a particulartheory, it is believed, as depicted in FIG. 1B, that the mixture spraysfrom nozzle 10 under low shear stress to form relatively large droplets11 of mixture, the large droplets expand and/or effervesce in explosionarea 12 to become a mist of fine suspension or solution droplets 13. Forexample, as shown in FIG. 1A, at distances from about 0 to about 2 cm,droplets can have an average size of about 400 μm that can be disruptedin the explosion area to a droplet size of about 10 μm only 3 cm fromthe nozzle orifice. Such ultrafine droplet production can also begenerated, e.g., by high-pressure conventional gases at pressures ofabout 1000 psi or greater (see, FIG. 2).

As will be appreciated by one of skill in the art, control of parameterssuch as particle size, size distribution, shape and form in theparticulate product will be dependent upon the operating conditions usedwhen carrying out the methods of the invention. Variables include theflow rate of the supercritical fluid, flow rate of the solution orsuspension, the concentrations of the bioactive material and excipients,diameter and length of the nozzle, the surface charge on the particles,and the relative humidity, temperature, and pressure inside the particleformation chamber and secondary drying chamber. For example, as shown inFIG. 3, the size of particles can be reduced with increased sprayingpressure. The histograms show that with high spraying pressure 30, theresultant particles averaged less than about 10 um, with a relativelynarrow population size range. With medium spray pressure 31 the averageparticle size was about 45 um, and with low spray pressure 32 theparticle size was about 200 um, both with relatively broad particlepopulation size ranges.

The flow rates of the high-pressure gas/near supercritical fluid and/orthe suspension/solution through the nozzle can be controlled to achievea desired particle size, size distribution, shape, and/or form. The flowrates can be established by adjusting independent valves in theconduits, which are preferably needle valves. Flow rates can also becontrolled by altering pumping conditions for the high-pressure gas/nearsupercritical fluid and/or the suspension/solution. Droplets in theinvention are typically produced with an average size ranging from about1 um to about 50 um, or about 5 um, before drying into particles.

Near supercritical fluid is typically introduced into the mixing chamberat a near the critical pressure of the fluid. High-pressure gas istypically introduced into the mixing chamber at a pressures above about1000 psi. The suspension or solution is typically introduced into themixing chamber at a flow rate from about 0.5 ml/min to about 50 ml/min,or about 3 ml/min (for a 100 um capillary restrictor) to about 30ml/min, and at a pressure near the pressure of the supercritical fluid.The mass flow ratio (gas/liquid) of the high-pressure gas or nearsupercritical fluid flow rate to the suspension or solution flow ratecan be between about 0.1 and 100, preferably between 1 and 20, morepreferably between 1 and 10, and most preferably around 5. Higherproportions and higher flow rates of suspension or solution can increasethe size of the droplets and the dry particles. Dry powder particles inthe invention can be controlled to have an average diameter, e.g., lessthan about 200 um, from about 0.5 um to about 150 um, typically fromabout 1 um to about 15 um; preferably, from about 3 um to about 10 um;and most preferably, from about 5 um to about 10 um, (see, FIG. 4).Droplet sizes (measured as the mass median diameter—MMD) can becontrolled to have a range from about 1 um to 400 um, from about 1 um toabout 200 um; preferably from about 5 um to about 50 um; and mostpreferably from about 3 um to about 10 um.

Pressurized gases that are suitable for spraying solutions orsuspensions of the invention include, e.g. nitrogen, carbon dioxide,oxygen, propane, nitrous oxide, helium, hydrogen, and/or the like; atpressures ranging from about 100 pounds per square inch (psi) to about15,000 psi. A number of fluids suitable for use as supercritical fluidsare known to the art, including, e.g., carbon dioxide, sulfurhexafluoride, chlorofluorocarbons, fluorocarbons, nitrous oxide, xenon,propane, n-pentane, ethanol, nitrogen, water, other fluids known to theart, and mixtures thereof. The supercritical fluid is preferably carbondioxide or mixtures of carbon dioxide with another gas such asfluoroform, and/or modifiers, such as ethanol. The temperature ofpressurized gases and/or supercritical fluids mixed with suspensions orsolutions in the methods can be, e.g., from about 0° C. to about 60° C.In a typical embodiment, the near supercritical fluid is CO₂ at apressure of about 1000 psi. Fine particles can also be dispersed underlower carbon dioxide pressures, e.g., 500, 750 and 950, (undernear-critical conditions). Near-critical fluids are defined (King, M.B., and Bott, T. R., eds. (1993), “Extraction of Natural Products usingNear-Critical solvents,” (Blackie Acad & Prof., Glasgow) pp. 1-33) assubstances maintained at pressures between 0.9 and 1.0 of their criticalpressure and/or temperature (in degree Kelvin).

The supercritical fluid can optionally contain one or more modifiers,for example, but not limited to, methanol, ethanol, isopropanol, and/oracetone. When used, the modifier preferably constitutes not more than20%, and more preferably constitutes between 1 and 10%, of the volume ofthe supercritical fluid. The term “modifier” is well known to thosepersons skilled in the art. A modifier (or co-solvent) may be describedas a chemical which, when added to a supercritical fluid, changes theintrinsic or colligative properties of the supercritical fluid in oraround its critical point.

Primary drying of the droplets can begin, e.g., during the expansion ofthe gas-liquid mixture. Primary drying can, e.g., convert liquiddroplets into primarily dried particles. Some of the solvent of thesuspension or solution can be dissolved in the near supercritical fluid,e.g., even before the expansion begins. As the spray expands, the fluidcan change state to a gas, removing latent heat and cooling the mist.The explosive expansion can break mixed droplets into smaller droplets.Degassing of high-pressure gases or supercritical fluids out of thedroplets can further disrupt them into finer droplets. The gasses andvapors around the fine droplets can be displaced by (i.e., be exchangedwith) a stream of drying gas flowing through the particle formationvessel. Significant amounts of solvent can be evaporated from the finedroplets on contact with the drying gasses; this can be accelerated bythe high surface to volume ratio of the droplets, a warm temperature ofthe drying gas, and a low relative humidity of the drying gas. Secondarydrying can take place in the particle formation vessel and/or the dryinggas can carry the fine droplets and/or primarily dried particles to asecondary drying chamber for further reduction of residual moisture.

Optionally, the fine mist of droplets can be sprayed into a stream ofcold fluid to freeze the droplets. The cold stream can be, e.g., a gas(e.g., CO₂), or a liquid (liquid nitrogen), at temperature between about−60° C. to about −200° C. The frozen droplets can be exposed to anenvironment of low pressure (i.e., a pressure less than atmospheric) toremove ice by sublimation to form, e.g., low density, lyophilized drypowder particles.

Secondary Drying

Secondary drying of the structurally stabilized and primarily driedparticles can, e.g., further remove entrapped solvent, residualmoisture, and/or water of molecular hydration, to provide a compositionof powder particles with significantly lower moisture content that isstable in storage, e.g., for extended periods at ambient temperatures.Secondary drying can involve, e.g., suspension of particles in a vortexof drying gas, suspension of particles in a fluidized bed of drying gas,and/or application of warm temperatures to the particles in a strongvacuum for several hours to days. The rapid drying and fine particlesizes formed during spraying and primary drying can allow reducedtemperatures and times for secondary drying in methods of the invention.

Secondary drying conditions can be used, e.g., to further lower themoisture content of particles. Particles can be collected in a secondarydrying chamber and held at a temperature below the glass transitiontemperature (See FIG. 5) of the dried (<1% moisture) formulation, orbetween about 5° C. and about 90° C., or between about 25° C. and about65° C., or about 35° C. The chamber can maintain a reduced pressure andsecondary drying can continue, e.g., for about 2 hours to about 5 days,or about 4 hours to about 48 hours, until residual moisture is reducedto a desired level. Secondary drying can be accelerated by providing anupdraft of drying gasses in the chamber to create a fluidized bedsuspension of the powder particles. Particles with lower residualmoisture generally show better stability in storage with time. Secondarydrying can continue until the residual moisture of the powder particlesis between about 0.5 percent and about 10 percent, or less than about 5percent. At very low residual moisture values, some bioactive moleculescan be denatured by loss of water molecules of hydration. Thisdenaturation can often be mitigated by providing alternative hydrogenbinding molecules, such as sugars, polyols, and/or polymers, in theprocess suspension or solution.

Because of the increased efficiency of the apparatus and methoddescribed herein, drying can be achieved at relatively low temperaturescompared to commonly used methods. Moreover, it has been found thatduring the adiabatic expansion, the temperature of the mixturedecreases, i.e., the net temperature around the resulting droplets islower because of self cooling. The temperature of the gas in theparticle formation vessel and the particle collector can be maintainedat or below the T_(g) of the dried powder particle or the denaturationtemperature of the biologically active material, and typically is aboutor less than about 90° C.; preferably, between about 25 and about 80°C.; and more preferably, between about 30 and about 50° C., or about 35°C. The reduced drying temperature can minimize activity loss from thedrying process and contribute to the enhanced biological activity whichis preserved in the dried fine particles recovered from the process.

The drying gas can be recycled and conditioned to provide desired dryingconditions. The drying gas can be a substantially inert gas, such asnitrogen, to avoid chemical degradation of the bioactive material duringdrying. The gas can be cycled from the particle formation vessel and/orsecondary drying chamber, through desiccators or condensers to removehumidity, through heat exchangers to heat or cool the gas to provide thedesired drying temperature, and recycled, e.g., back to the particleformation chamber. An ion generator can inject ions into the stream ofparticles to reduce charge build up and/or to control the agglomerationrate of fine particles into larger particle sizes.

Powder particles of the invention can have a size on drying, e.g.,suitable to the handling, reconstitution, and/or administrationrequirements of the product. For example, powder particles of bioactivematerials for administration by intranasal delivery by inhalation can belarger, at between about 20 um to about 150 um, than for deep pulmonarydelivery by inhalation, at between about 0.1 um to about 10 um. Theparticle size for products that reconstitute slowly can be smaller tospeed dissolution of the particles. Spray freeze-dried particles canhave, e.g., a lower density, because ice can be removed from dropletswithout collapse of a cake structure supported by the remaining solids.Such particles can have, e.g., a physically larger size for inhaledadministration due to their lower aerodynamic radius. Under some processconditions, the powder particles in the invention can have a hollowedhemispherical shape (see FIG. 6). Freeze-dried particles can, e.g., belarger than particles dried from liquid droplets and still retain quickreconstitution properties due to the porous nature of freeze-driedparticles. Powder particles of the invention can have average physicaldiameters, e.g., between about 0.1 um and about 200 um, between about 1um and about 50 um, or between about 2 um and about 20 um (See FIG. 4).

During the secondary drying process, e.g., a spray coat or otherprotective coating can be applied to the particles. For example, a mistof a polymer solution can be sprayed into a suspension of dryingparticles in a vortex or fluidized bed.

The methods of the invention result in a pharmaceutically-acceptable,powder particles comprising, e.g., at least one biologically activematerial within the amorphous glassy matrix. Preferably, the compositionis almost completely dry. Some water or other aqueous solvent can remainin the composition but typically, not more than about 5% residualmoisture remains by weight. The drying temperature can range from lessthan about 90° C., between about 25° C. and about 80° C., between about30° C. and about 50° C., or about 35° C. A typical secondary dryingprocess can include, e.g., raising the temperature to a dryingtemperature of from about 30° C. to about 55° C., and holding for fromabout 0.5 days to about 5 days to provide a stable dried powdercomposition with 0.1% to about 5%, or about 3% residual moisture. Asused herein, “dry”, “dried”, and “substantially dried” encompass thosecompositions with from about 0% to about 5% water. Preferably, thepowder matrix will have a moisture content from about 0.1% to about 3%as measured using the Karl Fisher method.

The resulting product can be an amorphous solid (see, X-raycrystallography chart, FIG. 7), wherein the glassy excipient material,e.g. sucrose, is in an amorphous glassy state and encases thebiologically active material, thereby substantially restrictingmolecular mobility and preventing protein unfolding. Without being boundto any particular theory, this process has been postulated to occureither via mechanical immobilization of the protein or the activeingredient by the amorphous glass or via hydrogen bonding to polar andcharged groups on the protein, i.e. via water replacement, therebypreventing drying induced denaturation and inhibiting further unwantedor degradative interactions. The glassy matrix stabilization theory hasprovided a useful albeit simplified way of describing the generalphenomenon of biopreservation. However, data accumulated from theliterature in the recent years have suggested that in a number ofinstances, the glassy state is neither necessary nor sufficient for longterm stabilization. It is important to note that the mechanismsattributed to stabilization of biologicals can be multifactorial and notlimited to the amorphous nature of the powder matrix in which the activeingredient is encased. Stabilization under the process described herecan involve a number of factors including but not limited to the thermalhistory that the biomaterial is subjected to, the reduction inconformational mobility and flexibility of the protein side chainsand/or reduction in the free volume as a result of the encasement,improvement in the structural rigidity of the matrix, reduction in thephase separation of excipient from the active ingredient, improvement inthe degree of water displacement by selecting the optimal hydrogenbonding donor. The latter is a function of the affinity and avidity ofthe excipient for the surface of the protein, nucleic acids,carbohydrate, or lipids being stabilized. In general, as long as thesolid is at a temperature below its glass transition temperature and theresidual moisture remaining in the excipients is relatively low, thelabile proteins and/or bioactive material containing lipid membranes canremain relatively stable.

Recovery of Bioactive Material in Particles

Powder particles of the invention are recovered with desired activityand in a form suitable to the intended route of administration. Powderparticles of the invention can be physically recovered from the processstream, e.g., by settling or filtration after drying. The methods of theinvention can provide high recovery of active and stable material due tothe moderate process conditions involved. Methods of the invention canprovide, e.g., particles adapted for administration as a highconcentration solution, an aerosol mist, intranasal deposited particles,or pulmonary deposited particles.

Physical recovery of powder particles can depend, e.g., on the amount ofmaterial retained or expelled by the spray-drying equipment, and lossesincurred due to particle size selection methods. For example, processmaterial containing the bioactive material can be lost in the plumbing,and on surfaces of the spray-drying equipment. Solutions or particlescan be lost in the process, e.g., when an agglomeration of spraydroplets grows and falls out of the process stream, or when under sizeddroplets dry to minute particles that are carried by drying gassesthrough the secondary drying chamber in a process waste stream. Processyields (the percent recovery of input active material through theprocess) of the invention can range, e.g., from more than about 70percent, or about 80 percent to about 98 percent, or about 90 percent.

Particles of a desired average size and size range, can be selected,e.g., by filtration, settling, impact adsorption, and/or other meansknown in the art. Particles can be sized by screening them through oneor more filters with uniform pore sizes. Large particles can byseparated by allowing them to fall from a suspension of particles in amoving stream of liquid or gas. Large particles can also stick byinertial impact to surfaces at the outside of a turning fluid streamwhile the stream carries away smaller or less dense particles. Smallerparticles can be separated by allowing them to be swept away in a streamof liquid or gas moving at a rate at which larger particles settle.

Recovery of active bioactive material can be affected, e.g., by physicallosses, cell disruption, denaturation, aggregation, fragmentation,oxidation, and/or the like, experienced during the spray-dry process.The recovery of bioactive material activity in the process is theproduct of the physical recovery times the specific activity ofrecovered material. The difference between the input activity and therecovered activity is sometimes referred to as “process loss”. Themethods of the invention offer reduced process loss, e.g., by convertingmore of the bioactive material into power particles that meet processspecifications. The methods of the invention also offer improvedspecific activity (active bioactive material/inactive bioactivematerial) in powder particle final product over the prior art, e.g., byproviding spray dry techniques that reduce shear stress, reduce dryingtime, reduce drying temperatures, and/or enhance stability. The specificactivity (e.g., ratio of an active protein or viable virus over thetotal protein or total virus particles) can remain relatively constantthrough the particle formation processes of the invention. The change inspecific activity of bioactive agents through the process can be, e.g.,less than about 2%, less than about 10%, less than about 30%, or lessthan about 50%.

Administration of Bioactive Materials

Where it is appropriate, the bioactive material of the invention can beadministered, e.g., to a mammal. Bioactive materials of the inventioncan include, e.g., peptides, polypeptides, proteins, viruses, bacteria,antibodies, cells, liposomes, and/or the like. Such materials can act astherapeutics, nutrients, vaccines, pharmaceuticals, prophylactics,and/or the like, that can provide benefits on administration to apatient, e.g., by gastrointestinal absorption, topical application,inhalation, and/or injection.

The bioactive material can be administered to a patient by topicalapplication. For example, the powder particles can be mixed directlyinto a salve, carrier ointment, pressurized liquid, gaseous propellants,and/or penetrant, for application to the skin of a patient. Alternately,the powder particles can, e.g., be reconstituted in an aqueous solventbefore admixture with other ingredients before application.

Bioactive materials of the invention can be administered by inhalation.Dry powder particles less than about 10 um in aerodynamic diameter canbe inhaled into the lungs for pulmonary administration. Optionally,powder particles of about 20 um, or greater, in aerodynamic diameter canbe administered intranasally, or to the upper respiratory tract, wherethey are removed from the air stream by inertial impact onto the mucusmembranes of the patient. The powder particles can alternately bereconstituted to a suspension or solution for inhalation administrationas an aqueous mist.

Bioactive materials of the invention can be administered by injection.The powder particles can be administered directly under the skin of apatient using, e.g., a jet of high pressure air. More commonly, thepowder particles can be, e.g., reconstituted with a sterile aqueousbuffer for injection through a hollow syringe needle. Such injectionscan be, e.g., intramuscular, intra venous, subcutaneous, intrathecal,intraperitoneal, and the like, as appropriate. Powder particles of theinvention can be reconstituted to a solution or suspension with abioactive material concentration, e.g., from less than about 0.1 ng/mlto from less than about 1 mg/ml to about 500 mg/ml, or from about 5mg/ml to about 400 mg/ml, as appropriate to the dosage and handlingconsiderations. Reconstituted powder particles can be further diluted,e.g., for multiple vaccinations, administration through IV infusion, andthe like.

The appropriate dosage (“therapeutically effective amount”) of thebiologically active material will depend, for example, on the conditionto be treated, the severity and course of the condition, whether thebiologically active material is administered for preventive ortherapeutic purposes, previous therapy, the patient's clinical historyand response to the biologically active material, the type ofbiologically active material used, and the discretion of the attendingphysician. The biologically active material is suitably administered tothe patent at one time, or over a series of treatments, and may beadministered to the patent at any time from diagnosis onwards. Thebiologically active material may be administered as the sole treatmentor in conjunction with other drugs or therapies useful in treating thecondition in question.

As a general proposition, the therapeutically effective amount of thebiologically active material administered will be in the range of about0.00001 (e.g., in the case of a live attenuated virus vaccine) to about50 mg/kg of patent body weight whether by one or more administrations,with the typical range of protein used being from less than about 0.01ng/kg to about 20 mg/kg, more preferably about 0.1 mg/kg to about 15mg/kg, administered daily, for example. However, other dosage regimensmay be useful. The progress of this therapy is easily monitored byconventional techniques.

The invention also encompasses methods of increasing the “shelf-life” orstorage stability of dried biologically active materials stored atelevated temperatures. Increased storage stability can be determined byrecovery of biological activity in accelerated aging trials. The dryparticle compositions produced by methods of the invention can be storedat any suitable temperature. Preferably, the compositions are stored atabout 0° C. to about 80° C. More preferably, the compositions are storedat about 20° C. to about 60° C. Most preferably, the compositions arestored at ambient temperatures.

COMPOSITIONS OF THE INVENTION

Compositions of the invention include, e.g., formulations for thesuspensions and solutions used in the process methods of the invention,and the stable powder particle products of bioactive materials preservedin a matrix with polyols and/or other excipients. The compositions canbe, e.g., suspensions or solutions suitable for spraying (liquid feedmaterial) with high-pressure gas or with a near supercritical fluid toprovide dry particles with improved stability. The suspensions orsolutions of bioactive material can comprise, e.g., a polyol, a polymer,and/or a surfactant.

Suspensions or Solutions for Spraying of Dry Powder Particles

Formulations for preparation of dry powder particle compositions of theinvention can include, e.g., bioactive materials, polymers, amino acids,polyols, surfactants, and/or buffers. Such formulations can be processedaccording to methods of the invention to provide stable compositions forstorage and administration of the bioactive materials. For example, acomposition of the invention can be an aqueous suspension of influenzavirus with sucrose, HES, and block copolymers of polyethylene andpolypropylene glycol (Pluronic), for spray drying with nearsupercritical carbon dioxide.

Bioactive materials of the invention include, e.g., materials withdetectable bioactivity in living systems, biological cells and moleculesused in analysis, biological cells and molecules used in medicine,biological cells and molecules used in research, and/or the like. Forexample, bioactive materials of the compositions of the inventioninclude proteins, peptides, nucleic acids, bacteria, cells, antibodies,enzymes, serums, vaccines, liposomes, viruses, and/or the like.

Bioactive materials in the powder particles of the invention can be,e.g., highly pure and active at the time of drying the powder particles,due to the reduced shear stress, the low drying temperatures, and theshort drying times used in their preparation. Bioactive materials are,e.g., stable in the powder particles due to the low initial processdegradation and the protective aspects of the composition excipients.Bioactive materials of the composition can be, e.g., reconstituted athigh concentrations without degradation due to the high surface tovolume ratio of the particles and the solubility enhancements providedby the excipients of the composition.

Solutions or suspensions spray-dried to form the powder particles of theinvention contain, e.g., the bioactive materials of the invention in anamount ranging from less than about 0.1 ng/ml to about 200 mg/ml, fromless than about 0.5 mg/ml to about 150 mg/ml, from about 10 mg/ml toabout 80 mg/ml, or about 50 mg/ml. Bioactive materials in the dry powderparticles of the invention are generally present in amounts ranging,e.g., from less than about 0.01 weight percent to about 80 weightpercent, from about 40 weight percent to about 60 weight percent, orabout 50 weight percent. Bioactive materials of the reconstitutedcomposition can be present in concentrations generally ranging, e.g.,from less than about 0.1 ng/ml to about 500 mg/ml, from about 0.5 mg/mlto about 400 mg/ml, or about 1 mg/ml.

Bioactive materials can include complex materials with lipid membranes,such as, e.g., biologically active, viable or non-living, cells,viruses, and/or liposomes. For example the bioactive materials caninclude vaccines, viruses, liposomes, bacteria, platelets, and cells.Viral bioactive agents can include, e.g., influenza virus, parainfluenzavirus, human metapneumovirus, respiratory syncytial virus, herpessimplex virus, SARS virus, corona virus family members, cytomegalovirus,and/or Epstein-Barr virus which can be present in the suspensions orsolutions of the invention in amounts ranging from about 10³ TCID₅₀/mLto about 10¹² TCID₅₀/mL, or about 10⁶ TCID₅₀/mL. Viral bioactivematerials will generally be present in the suspensions or solutions inan amount of less than about 1%; more preferably, less than about0.001%; and most preferably, less than about 0.0001% by weight. Driedpowder particle compositions of the invention can provide virus presentin an amount, e.g., from about 10¹ TCID₅₀/g to not more than about 10¹²TCID₅₀/g. Dried powder particle compositions can provide virus presentin an amount, e.g., of about 10¹ TCID₅₀/g, about 10² TCID₅₀/g, about 10³TCID₅₀/g, about 10⁴ TCID₅₀/g, about 10⁵ TCID₅₀/g, about 10⁶TCID₅₀/g,about 10⁷ TCID₅₀/g, about 10⁸ TCID₅₀/g, about 10⁹ TCID₅₀/g, about 10¹⁰TCID₅₀/g, or about 10¹¹ TCID₅₀/g.

Polyols of the invention can include, e.g., various sugars,carbohydrates, and alcohols. For example, the polyols can includenon-reducing sugars, sucrose, trehalose, sorbose, melezitose, and/orraffinose. The polyols can include, e.g., mannose, maltose, lactose,arabinose, xylose, ribose, rhamnose, galactose and glucose, mannitol,xylitol, erythritol, threitol, sorbitol, glycerol, L-gluconate, and/orthe like. Where it is desired that the formulation be freeze-thawstable, the polyol is preferably one which does not crystallize atfreezing temperatures (e.g. −20° C.) such that it destabilizes thebiologically active material in the formulation. The amount of polyolused in the suspension or solution can vary depending on the nature ofthe bioactive material, the type of polyol, and the intended use.However, generally, the final concentration of polyol is between about0.1% and 40%; more preferably, between about 1% and 20% by weight. In aparticularly preferred embodiment, the suspension or solution comprisesabout 10% sucrose.

Polymers of the invention can include, e.g., various carbohydrates,polypeptides, linear and branched chain hydrophilic molecules. Forexample, polymers of the formulation can include oxidized starch,carboxymethyl starch and hydroxyethyl starch (HES), dextran,non-recombinant human serum albumin (HSA), as well as nonhydrolyzed andhydrolyzed gelatin, gelatin, ovalbumin, collagen, chondroitin sulfate, asialated polysaccharide, actin, myosin, microtubules, dynein, kinetin,alginate, and/or the like. These additives do not necessarily solelystabilize the biologically active material against inactivation; theyalso may help to prevent the physical collapse of the spray driedmaterial during primary drying, lyophilization, secondary drying, and/orsubsequent storage in the solid state. Preferably, HES is used with amolecular weight of between about 100,000 and 300,000; and morepreferably, about 200,000. Generally, the concentration of HES can befrom about 0.5% to about 10%; more preferably, between about 1 and 5% ofthe suspension or solution by weight. A preferred formulation comprisesabout 5% HES.

Suspensions or solutions for preparation of the compositions of theinvention can include, e.g., one or more surfactants to aid insolubility and stability of formulation constituents. Surfactants can bepresent in formulations of the invention in a concentration ranging fromabout 0.001 weight percent to about 5 weight percent, or about 0.01weight percent to about 1 weight percent. The surfactants can include,e.g., nonionic detergents, such as polyethylene glycol sorbitanmonolaurate (Tween 20), polyoxyethylenesorbitan monooleate (Tween 80),block copolymers of polyethylene and polypropylene glycol (Pluronic),and/or the like.

Examples of suitable non-ionic surfactants are alkylphenyl alkoxylates,alcohol alkoxylates, fatty amine alkoxylates, polyoxyethylene glycerolfatty acid esters, castor oil alkoxylates, fatty acid alkoxylates, fattyacid amide alkoxylates, fatty acid polydiethanolamides, lanolinethoxylates, fatty acid polyglycol esters, isotridecyl alcohol, fattyacid amides, methylcellulose, fatty acid esters, silicone oils, alkylpolyglycosides, glycerol fatty acid esters, polyethylene glycol,polypropylene glycol, polyethylene glycol/polypropylene glycol blockcopolymers, polyethylene glycol alkyl ethers, polypropylene glycol alkylethers, polyethylene glycol/polypropylene glycol ether block copolymersand mixtures of these, polyacrylates and acrylic acid graft copolymers.Other nonionic surfactants are known per se to those skilled in the artand have been described in the literature. Preferred substances arepolyethylene glycol, polypropylene glycol, polyethyleneglycol/polypropylene glycol block copolymers, polyethylene glycol alkylethers, polypropylene glycol alkyl ethers, polyethyleneglycol/polypropylene glycol ether block copolymers and mixtures ofthese. Particularly preferred surfactants include polymers of a mixtureof polyoxyethylene and polyoxypropylene such as Pluronic F68 (availablefrom BASF).

Examples of suitable ionic surfactants are alkylarylsulfonates,phenylsulfonates, alkyl sulfates, alkyl sulfonates, alkyl ethersulfates, alkyl aryl ether sulfates, alkyl polyglycol ether phosphates,polyaryl phenyl ether phosphates, alkylsulfosuccinates, olefinsulfonates, paraffin sulfonates, petroleumsulfonates, taurides,sarcosides, fatty acids, alkylnaphthalenesulfonic acids,naphthalenesulfonic acids, lignosulfonic acids, condensates ofsulfonated naphthalenes with formaldehyde or with formaldehyde andphenol and, if appropriate, urea, lignin-sulfite waste liquor, includingtheir alkali metal, alkaline earth metal, ammonium and amine salts,alkyl phosphates, quaternary ammonium compounds, amine oxides, betainesand mixtures of these. Preferred substances include Pluronic F68 orPluronic F188 with polyoxyethylene sorbitan monolaurate (i.e., Tween 20,available from Sigma) being particularly preferred.

Amino acid excipients can be present, e.g., to enhance stability,control the pH, affect reconstitution rate, serve as bulking agents,scavenge oxidizing molecules, etc. Examples of suitable amino acids arearginine, lysine, methionine, histidine, glycine, glutamic acid, and/orthe like.

Buffers can be present, e.g., to control pH, enhance stability, affectconstituent solubility, provide comfort on administration, and the like,in formulations for preparation of dry powder compositions. FormulationpH can be controlled in the range of about pH 3 to about pH 10, fromabout pH 6 to about pH 8, or about pH 7.2. Preferred buffers are oftenpaired acid and salt forms of a buffer anion generally recognized assafe for the particular route of administration of the bioactivematerial. Typical buffers for use in the formulations and compositionsof the invention include, e.g., amino acids, potassium phosphate, sodiumphosphate, sodium acetate, sodium citrate, sodium, succinate, ammoniumbicarbonate, carbonates, and the like. Generally, buffers are used atmolarities from about 1 mM to about 2 M, with from about 2 mM to about 1M being preferred, and from about 10 mM to about 0.5 M being especiallypreferred, and 25 to 50 mM being particularly preferred.

Exemplary formulations for suspensions with influenza virus include thefollowing. Influenza virus in aqueous suspension with 10% sucrose, 5%hydroxyethyl starch (Fesnius), 2 mM methionine, 50 mM KH2PO4 buffer (pH7.2), and 0.1% Pluronic F-68. Influenza virus in aqueous suspension with10% sucrose, 5% hydroxyethyl starch, 75 mM KH2PO4 buffer (pH 7.2), 2 mMmethionine, and 0.01% Pluronic F-68. Influenza virus in aqueoussuspension with 5% sucrose, 2% trehalose, 0.2% Pluronic-F68; 10 mMmethionine, 2% arginine, 2 mM EDTA, and 50 mM KH2PO4 buffer (pH 7.2).

In one embodiment, the formulation contains the above-identified agents(i.e., biologically active material, polyol, surfactant, and gelatin)and is essentially free of one or more preservatives, such as benzylalcohol, phenoly, m-cresol, chlorobutanol, and benethonium chloride). Inanother embodiment, a preservative may be included in the formulation,particularly when the formulation is a multidose formulation.

One or more pharmaceutically acceptable carriers, excipients, orstabilizers such as those described in Remington's PharmaceuticalSciences 16^(th) Edition, Osol, A. Ed. (1980) may be included in theformulation provided that they do not adversely affect the desiredcharacteristics of the formulation. Acceptable carriers, excipients orstabilizers are nontoxic to recipients at the dosages and concentrationsemployed and include; additional buffering agents; co-solvents;salt-forming counterions such as potassium and sodium; antioxidants,such as methionine, N-acetyl cysteine, or ascorbic acid; chelatingagents, such as EDTA or EGTA. Amino acids, such as, e.g., arginine andmethionine can be included in the formulations. Arginine can be presentin the formulations in an amount ranging from about 0.1 weight percentto about 5 weight percent. Methionine can be present in the formulationin a concentration ranging from about 1 mM to about 50 mM or about 10mM. Glycerol can be present in the formulation in a concentrationranging, e.g., from about 0.1 weight percent to about 5 weight percent,or about 1 weight percent. EDTA can be present in the formulation in aconcentration ranging, e.g., from about 1 mM to about 10 mM, or about 5mM.

The present invention includes articles of manufacture comprising acontainer containing dried powder particles prepared by spray drying amixture of pressurized gas or near supercritical gas with a suspensionor solution of bioactive material, a polyol, a polymer additive, and asurfactant. In an embodiment of the invention, an article of manufactureis provided comprising a container which holds the pharmaceuticalformulation of the present invention and optionally providesinstructions for its use. Suitable containers include, for example,bottles, vials, blister packs, and syringes. The container can be formedfrom a variety of materials such as glass or plastic. An exemplarycontainer is a 3-20 cc single use glass vial. Alternatively, for amultidose formulation, the container may be 3-100 cc glass vial. Thecontainer holds the formulation and the label on, or associated with,the container may indicate directions for use. The article ofmanufacture may further include other materials desirable from acommercial and user standpoint, including other buffers, diluents,filters, needles, syringes, and package inserts with instructions foruse.

The powder particles described herein are stable, i.e., they retaintheir biological activity and are chemically and/or physically stable.The powder particles were tested for stability by subjecting them toaging at elevated temperature (e.g., 37° C.) and measuring theirbiological activity, chemical and/or physical stability. Results ofthese studies demonstrate that these particles which were dried at 55°C. using the methods of the invention were stable for at least ninemonths at 25° C. (see, FIG. 8). Particles which were dried at 35° C.were stable for at least about 13 months at 25° C. and for 2 years ormore at 4° C. Such powder particles are stable even when highconcentrations of the biologically active material are used. Thus, thesedry particles are advantageous in that they may be shipped and stored attemperatures at or above room temperature for long periods of time.

Apparatus of the Invention

The apparatus of the invention can include, e.g., a container (firstchamber) to hold the suspension or solution, a pressure vessel (secondchamber) to hold a high-pressure gas and/or near supercritical fluid,conduits with control valves to control flow from the first and secondchambers into a mixing chamber, a nozzle with a capillary restrictorthrough which a mixture can be sprayed into a particle formation vessel,and a flow of drying gas that can provide primary and/or secondarydrying of particles from the particle formation vessel. Secondary dryingof particles can include, e.g., settling to a warm surface in a vacuum,lyophilization of frozen particles, suspension in a vortex of dryinggas, and/or suspension in a fluidized bed of drying gas.

As shown, for example, in FIG. 9, the apparatus can comprise, e.g., aspray nozzle directing a mist of droplets into a spray dryer. A virussuspension in first chamber 30 can be pumped by HPLC pump 31 throughfirst conduit 32 to a T-intersection and into mixing chamber 33 of spraynozzle 34. A pressurized gas or near supercritical CO₂ fluid in secondchamber 35 can be pumped by high pressure pump 36 capable of providing aselected pressure (e.g., from about 250-15000 psi) through secondconduit 37 to the T-intersection to mix with the suspension in themixing chamber. The mixture can be ejected from the mixing chamberthrough capillary restrictor 38 to form a spray mist of fine dropletsthat dry into particles in particle formation chamber 39. A drying gas,driven by fan 40, can displace gas and solvent vapors from the spray toprovide primary drying to the particles while, e.g., carrying them tosecondary drying chamber 41. Primarily dried particles from the particleformation chamber can experience secondary drying by contact with thedrying gas before and after settling into particle collection vessel 42.The spray nozzle can be adapted to function with a variety of spraydryers and can be scaled to accommodate processes spraying up to severalliters per hour. Spray dryer components of the apparatus can be adaptedfrom, e.g., lab bench spray dryers made by Buchi (BrinkmannInstruments).

Certain chambers and vessels of the apparatus can have multiple oralternate functions to carry out the methods of the invention. Forexample, in some embodiments, the particle formation vessel can also actas a secondary drying chamber, and/or a particle collection vessel.Optionally, the secondary drying chamber can comprise a vortex chamber,fluidized bed chamber, a particle sizing chamber, a polymer coatingchamber, and/or a particle collection vessel.

Fluids and Gasses

The apparatus of the invention can have chambers and conduit to hold andtransfer the high-pressure gas or near supercritical fluids, andsuspensions or solutions, to a mixing chamber. The sprayed droplets canexperience primary and secondary drying, e.g., by contact with dryinggases.

The high-pressure gases and/or near supercritical fluids can be thosedescribed in the Methods section and Compositions section above, such asnitrogen, carbon dioxide, sulfur hexafluoride, chlorofluorocarbons,fluorocarbons, nitrous oxide, xenon, propane, n-pentane, ethanol,nitrogen, water, and/or the like. Modifiers, such as certain alcoholscan be dissolved in the supercritical fluids to, e.g., adjust thesolvent, critical point and/or expansion properties of the fluid.

The suspensions or solutions can include a bioactive material and apolyol. Exemplary bioactive materials include proteins, peptides,nucleic acids, bacteria, cells, antibodies, enzymes, serums, vaccines,liposomes, and viruses. Polyols in the suspensions or solutions of theapparatus include, e.g., trehalose, sucrose, sorbose, melezitose,glycerol, fructose, mannose, maltose, lactose, arabinose, xylose,ribose, rhamnose, palactose, glucose, mannitol, xylitol, erythritol,threitol, sorbitol, and raffinose.

The suspensions or solutions of the apparatus can include additionalexcipients, such as polymers and surfactants. The polymers can be, e.g.,starch, starch derivatives, carboxymethyl starch, hydroxyethyl starch(HES), dextran, human serum albumin (HSA), and/or gelatin. Thesurfactants can be, e.g., polyethylene glycol sorbitan monolaurate,polyoxyethylenesorbitan monooleate, block copolymers of polyethyleneand/or polypropylene glycol.

Apparatus Hardware

The apparatus of the invention can include, e.g., a first chamber tohold a suspension or solution, a second chamber to hold a high-pressuregas and/or near supercritical fluid, a nozzle with a mixing chamber anda capillary restrictor with an outlet orifice, a particle formationchamber, and a secondary drying chamber. Suspension or solution can bepumped into the mixing chamber under pressure through a first conduit tomix with near supercritical fluid pumped into the mixing chamber througha second conduit. The mixture can spray out of the nozzle as a mist intothe particle formation chamber where it can begin to dry on contact witha stream of drying gas. Secondary drying can take place by contact withwarmed chamber walls and/or by contact with the stream of drying gas inthe particle formation vessel and/or a secondary drying chamber.

In a preferred embodiment, the particle formation vessel and/orsecondary drying chamber are housed within an environmental controlchamber. The controlled humidity and temperature of the environmentalcontrol chamber can be the source of drying gases. Inlet gas from theenvironmental control chamber to the particle formation vessel can bemixed with droplets emitted from the capillary constrictor as a finemist. The fine mist can be partially dried (i.e., from a droplet into aparticle) in the particle formation vessel before transfer in a streamof drying gas to a secondary drying chamber, such as a cyclonic vortexchamber. The stream of drying gas can continue to a gas outlet port backinto a environmental control chamber where the gas can be reconditioned.The apparatus can further comprise a desiccant or condenser system forremoving moisture from the gas and/or the environmental control chamber.A heat exchanger can be used to control the temperature of the recycledgas and prevent excessive build up of temperature inside the environmentcontrolled chamber. Typically, the chamber is cooled by introduction ofliquid nitrogen from a liquid nitrogen reservoir with control by anoptional temperature controller which can automatically meter the liquidnitrogen to provide for a relatively invariant temperature inside theenvironmental control chamber. Optionally, the environmental controlchamber can be cooled by a refrigeration heat exchanger (evaporator).The environmental control chamber is typically vented to the ambientroom pressure via a pressure control port which can be valved orpressure gated. Spray drying into a reduced moisture controlled gas canprovide a large moisture differential between the sprayed droplets andthe drying chamber environment. The effect can be a reduced input heatrequirement for the primary drying phase.

The first and second chambers can be pressurized, and/or pumps can beemployed in the conduits, to deliver high-pressure gas and/or nearsupercritical fluid, and/or suspensions or solutions, to the mixingchamber. The rate of delivery can be controlled by means commonlypracticed in the art, such as, e.g., by controlling the pumping rate orby controlling valves in the conduits. The pumps can be any type knownin the art, such as, e.g., peristaltic pumps, rotary pumps, diaphragmpumps, piston pumps, and the like. Valves can be any appropriate styleknown in the art, including, e.g., ball and seat, diaphragm, needle,that can restrict the flow of pressurized fluids. Typically, the secondcontainer is pressurized, refrigerated and/or insulated to hold thepressurized gas or fluid at near critical conditions.

The mixing chamber can be, e.g., an enlarged space between conduitinflow ports and the capillary restrictor output orifice. The conduitscan be generally directed to flow the pressurized gas and/or nearsupercritical fluid and suspensions or solutions into each other, toenhance mixing. The conduits can meet at a T-intersection with flowsmeeting head on, or at an intersection wherein flows meet at less than180 degrees opposition, e.g., wherein the first conduit and/or secondconduit direct flow at an angle less than 90 degrees from the axis offlow in the mixing chamber. Flows can meet indirectly, e.g., with anoffset, to create a swirling, vortex or turbulent flow, since this canpromote more thorough mixing and create more monodispersed gas-liquidemulsion, as is appreciated by those skilled in the art. The main bodyof the chamber can have a long aspect ratio to enhance contact surfacesbetween the supercritical fluid and suspensions or solutions. The mixingchamber can have passage configurations that include baffles, beads,channels, obstructions, constrictions, and/or the like, to enhancemixing of the high-pressure gas and/or supercritical fluid with thesuspensions or solutions. The mixing chamber can be a conduit with aninternal diameter greater than the internal diameter of the capillaryrestrictor. The mixing chamber can be a part of the nozzle, or aseparate component of the apparatus.

The capillary restrictor can be, e.g., a conduit that provides arestriction to fluid flow to help maintain a high pressure or nearsupercritical conditions in the mixing chamber. The capillary restrictorcan have, e.g., an outlet orifice through which the high-pressuregas/near supercritical fluid mixture with suspension or solution can besprayed. The size of the capillary restrictor internal diameter andoutlet orifice can affect the size of droplets produced in the spray;with larger droplets (and ultimately, particles) generally formed byspraying from, e.g., larger outlets. Typically, the capillary restrictorhas a length from about 2 inches to about 6 inches, and an internaldiameter and/or outlet diameter, e.g., of about 50 um or less, to about1000 um, from about 50 um to about 500 um, or about 100 um.

The mixture sprays out of the nozzle into a particle formation vesselwhere it, e.g., expands to gases and disrupted fluid feed droplets. Adrying gas can be introduced into the particle formation vessel todisplace mixture gasses (expanded gases and evaporated solvents) fromthe droplets. The drying gasses can contact the droplets to evaporateadditional solvent from them to form particles. The drying gasses cancarry droplets and/or particles to other chambers for processing by themethods of the invention. For example, primarily dry particles can besuspended in a stream of drying gas in the particle formation vessel, orbe carried to a separate chamber, for secondary drying, sizing, coating,and/or collection. The drying gas can be, e.g., an inert gas, such asnitrogen, at a temperature below the glass transition temperature of thepowder particles. The apparatus can include heat exchangers to controlthe temperature of the drying gas, e.g., less than about 90° C., betweenabout 25° C. to about 80° C., between about 30° C. and 50° C., or about35° C. Preferred drying gas (inlet gas) temperatures during particleformation in the methods of the invention are less than 65° C., orbetween about 30° C. and about 55° C., or about 35° C. The apparatus caninclude condensers or desiccators to lower the relative humidity, orsolvent level, of the drying gas, e.g., so it can be recycled or sent towaste without harm to the environment.

The particle formation vessel or a secondary drying vessel can beadapted to provide a cyclonic vortex chamber. Particles, carried in astream of drying gas, can, e.g., enter a long cylindrical or conicalchamber at one end through an offset port. The gases can swirl manytimes in a spiral route from the inlet end of the chamber to an outletend. Such a route can take considerable time with the particlesreceiving warmth from the gas and chamber walls while they continue tolose residual moisture.

The particle formation vessel or a secondary drying vessel can beadapted to provide a fluidized bed chamber. Particles suspended in thestream of drying gas can be transferred, e.g., to an inlet at the bottomof a cylindrical chamber where they can become suspended in an updraftof the drying gas. Optionally, particles can be collected at the bottomof a chamber before directing drying gas from below to suspend theparticles in a fluidized bed. The particles can remain suspended as afluidized bed for a considerable time while residual moisture continuesto be lost. Size separation can take place in the fluidized bed chamberas small particles are lost in the waste stream out the top of thechamber and large particles settle the bottom. Polymer coatings can beapplied to particles, e.g., by spraying a mist of polymer solution intothe fluidized bed to dry as a coat on the particles.

The particle formation vessel, or secondary drying vessel, can beadapted to provide a collection vessel for collection of dried powderparticles. For example, particles flowing in a transfer conduitsuspended in gas can be directed into a chamber with considerably largerdiameter than the transfer conduit. The velocity of the gas can slow inthe larger chamber allowing the particles to fall to the floor of thechamber while the gas exits to waste above. The particles can accumulatein a removable container at the floor of the chamber where they can berecovered for use, packaging, or storage.

The present invention includes kit comprising, e.g., elements of theapparatus and process materials facilitating practice of the methods ofthe invention. The kits of the invention can be a container containingan apparatus element of the invention, such as a vessel of pressurizedgas or near supercritical fluid, suspension or solution components (suchas bioactive material or process solutions of a polyol, a polymer, anamino acid, a surfactant, and/or a buffer), a spray nozzle, a collectionvessel, and/or the like, for use in practicing methods of preparingdried particle compositions of the invention. The kit can besubstantially sterilizable, e.g., made of materials tolerant of thetemperature and moisture in an autoclave, tolerant of ionizingradiation, and/or tolerant of radiation produced within a microwaveoven. The kits of the invention can include instructional materialsteaching the use of apparatus, apparatus elements, and/or processmaterials of the invention to prepare dry particles of bioactivematerials.

EXAMPLES

The following examples are offered to illustrate, but not to limit theclaimed invention.

Example 1 Formulations for Spraying Influenza Suspensions

Formulations such as those shown below were prepared according to themethods of this invention using B/Harbin influenza virus or placebo. pHwas adjusted with either sodium hydroxide or potassium hydroxide. Usefulformulations for spray drying attenuated Influenza viruses can include,e.g., about 10% to about 2% trehalose, about 40% to about 5% sucrose,about 1% sorbitol, about 5% to about 2% HES, about 2% ovalbumin, about5% to about 2% gelatin, about 1% PVP, about 2% to about 0.01% PluronicF68, about 0.03% Tween 20, about 10 mM to about 2 mM methionine, about5% to about 0.5% arginine, about 23 mM EDTA, about 0.5% to about 0.05%glycerol, about 10% to about 1% glutamate, and/or about 10 mMN-acetylcysteine.

Polymer Polyol Additive Surfactant Other AV020 5% trehalose 5% HES 0.01%Pluronic 75 mM, pH 7.2 KPO4 buffer; F68 2 mM methionine AV021 5%trehalose 5% HES 0.03% Tween 20 75 mM, pH 7.2 KPO4 buffer; 2 mMmethionine AV022 5% trehalose 5% HES 0.05% Pluronic 75 mM, pH 7.2 KPO4buffer; F68 2 mM methionine AV023a 10% sucrose 5% HES 0.01% Pluronic 75mM, pH 7.2 KPO4 buffer; F68 10 mM N-acetylcysteine AV023 10% sucrose 5%HES 0.01% Pluronic 75 mM, pH 7.2 KPO4 buffer; F68 2 mM methionine; 2 mMEDTA; 0.5% arginine AV024 10% sucrose 5% HES 0.01% Pluronic 75 mM, pH7.2 KPO4 buffer; F68 2 mM methionine; 1% PVP; 0.5% arginine AV025 5%sucrose; — 0.05% Pluronic 50 mM, pH 7.2 KPO4 buffer; 2% trehalose F68 2mM EDTA; 2% arginine AV026 5% sucrose; 2% HES 0.05% Pluronic 50 mM, pH7.2 KPO4 buffer; 2% trehalose F68 2 mM EDTA; 2% arginine AV027 5%sucrose; — 0.05% Pluronic 50 mM, pH 7.2 KPO4 buffer; 2% trehalose F68 10mM methionine; 2 mM EDTA; 2% arginine AV028 5% sucrose; 2% HES 0.05%Pluronic 50 mM, pH 7.2 KPO4 buffer; 2% trehalose F68 10 mM methionine; 2mM EDTA; 2% arginine AV029 5% sucrose; — 0.05% Pluronic 50 mM, pH 6.8KPO4 buffer; 2% trehalose F68 10 mM methionine; 2 mM EDTA; 2% arginineAV030 5% sucrose; 2% HES 0.05% Pluronic 50 mM, pH 7.2 KPO4 buffer; 2%trehalose F68 10 mM methionine; 2 mM EDTA; 2% arginine AV031 2%trehalose 2% HES 0.05% Pluronic 50 mM, pH 7.2 KPO4 buffer; F68 2 mMEDTA; 0.2% sodium thiosulphate; 2% arginine AV032 5% sucrose; 2% HES0.05% Pluronic 50 mM, pH 7.2 KPO4 buffer; 2% trehalose F68 10 mMmethionine; 2 mM EDTA; 2% arginine AV033 5% sucrose; 0.05% Pluronic 50mM, pH 7.2 KPO4 buffer; 2% trehalose F68 10 mM methionine; 2 mM EDTA; 2%arginine AV034 5% sucrose; 2% HES 50 mM, pH 7.2 KPO4 buffer; 2%trehalose 10 mM methionine; 2 mM EDTA; 2% arginine AV035 10% 100 mM, pH7.2 KPO4 sucrose; buffer; 5 mM TMAO; 10% trehalose AV036 10% 100 mM, pH7.2 KPO4 sucrose; buffer; 5 mM TMAO; 0.5% 10% glycerol trehalose AV03710% 100 mM, pH 7.2 KPO4 sucrose; buffer; 5 mM TMAO; 0.5% 10% glyceroltrehalose AV038 10% 100 mM, pH 7.2 KPO4 sucrose; buffer; 10 mM N- 10%acetylcysteine; 0.5% glycerol trehalose AV039 5% sucrose; 2% 0.05%Pluronic 50 mM, pH 7.2 KPO4 buffer; 2% trehalose ovalbumin F68 10 mMmethionine; 10 mM N-acetylcysteine; 2 mM EDTA; 2% arginine; AV040 5%sucrose; 2% 0.05% Pluronic 50 mM, pH 7.2 KPO4 buffer; 2% trehaloseovalbumin F68 10 mM methionine; 2 mM EDTA; 2% arginine; AV041 5%sucrose; 2% gelatin 0.05% Pluronic 50 mM, pH 7.2 KPO4 buffer; 2%trehalose (K&K) F68 10 mM methionine; 2 mM EDTA; 2% arginine AV042 5%sucrose; 0.05% Pluronic 50 mM, pH 7.2 KPO4 buffer; 2% trehalose F68 10mM methionine; 5 mM TMAO; AV043 5% sucrose; 2% Pluronic F68 50 mM, pH7.2 KPO4 buffer; 2% trehalose 10 mM methionine; 2 mM EDTA; 2% arginineAV044 5% sucrose; 0.05% Pluronic 50 mM, pH 7.2 KPO4 buffer; 2% trehaloseF68 2 mM EDTA; 2% arginine; 1% L-glutamate AV045 5% sucrose; 0.1%Pluronic 100 mM, pH 7.2 KPO4 2% trehalose F68 buffer; 10 mM N-acetylcysteine; 2 mM EDTA; 2% arginine AV046 5% sucrose; 0.1% Pluronic100 mM, pH 7.2 KPO4 2% trehalose F68 buffer; 10 mM N- acetylcysteine; 5mM TMAO; 2 mM EDTA; 2% arginine; 0.05% glycerol AV047 5% sucrose; 0.2%Pluronic 50 mM, pH 7.2 KPO4 buffer; 2% trehalose F68 10 mM methionine; 2mM EDTA; 2% arginine AV048 5% sucrose; 0.05% Pluronic 50 mM, pH 7.2citrate buffer; 2% trehalose F68 10 mM methionine; 2 mM EDTA; 2%arginine AV049 5% sucrose; 0.05% Pluronic 50 mM, pH 7.2 KPO4 buffer; 2%trehalose F68 10 mM methionine; 2 mM EDTA; 2% arginine AV050 5% sucrose;0.05% Pluronic 50 mM, pH 7.2 KPO4 buffer; 2% trehalose F68 10 mMmethionine; 2 mM EDTA; 2% arginine AV051 5% sucrose; 0.05% Pluronic 50mM, pH 7.2 KPO4 buffer; 2% trehalose F68 10 mM methionine; 2 mM EDTA; 2%arginine AV052 5% sucrose; 0.05% Pluronic 50 mM, pH 7.2 KPO4 buffer; 2%trehalose F68 10 mM methionine; 2 mM EDTA; 2% arginine AV053 5% sucrose;50 mM, pH 7.2 KPO4 buffer; 2% trehalose 10 mM methionine; 2 mM EDTA; 2%arginine AV054 5% sucrose; 2% HES 0.05% Pluronic 50 mM, pH 7.2 KPO4buffer; 2% trehalose F68 10 mM methionine; 2 mM EDTA; 2% arginine AV0555% sucrose; 0.05% Pluronic 50 mM, pH 7.2 KPO4 buffer; 2% trehalose F6810 mM methionine; 2 mM EDTA AV056 5% sucrose; 0.05% Pluronic 50 mM, pH7.2 KPO4 buffer; 2% trehalose F68 10 mM methionine; 2% arginine AV057 5%sucrose; 50 mM, pH 7.2 KPO4 buffer; 2% trehalose 10 mM methionine; 2 mMEDTA; 2% arginine AV058 2% trehalose 0.05% Pluronic 50 mM, pH 7.2 KPO4buffer; F68 10 mM methionine; 2 mM EDTA; 2% arginine AV059 5% sucrose0.05% Pluronic 50 mM, pH 7.2 KPO4 buffer; F68 10 mM methionine; 2 mMEDTA; 2% arginine AV060 5% sucrose; 0.05% Pluronic 50 mM, pH 7.2 KPO4buffer; 2% trehalose F68 2 mM EDTA; 2% arginine AV061 5% sucrose; 0.05%Pluronic 50 mM, pH 7.2 KPO4 buffer; 2% trehalose F68 10 mM methionine; 2mM EDTA; 2% arginine AV062 6% sucrose; 0.05% Pluronic 50 mM, pH 7.2 KPO4buffer; 1% sorbitol F68 10 mM methionine; 2 mM EDTA; 2% arginine AV0637% sucrose 0.05% Pluronic 50 mM, pH 7.2 KPO4 buffer; F68 10 mMmethionine; 2% arginine AV064 6% sucrose; 0.05% Pluronic 50 mM, pH 7.2KPO4 buffer; 1% sorbitol F68 10 mM methionine; 2 mM EDTA; 2% arginineAV065 10% 5% HES 0.2% Pluronic 50 mM, pH 7.2 KPO4 buffer; sucrose; 2%F68 10 mM methionine; 2 mM trehalose EDTA; 2% arginine AV066 5% sucrose;2% HES 50 mM, pH 7.2 KPO4 buffer; 2% trehalose 2% arginine AV067 10%sucrose 2% HES 50 mM, pH 7.2 KPO4 buffer; 5% arginine AV068 10% sucrose2% HES 50 mM, pH 7.2 KPO4 buffer; 1 mM ZnCl2; 5% arginine AV070 40%sucrose 5% gelatin 0.02% Pluronic 25 mM, pH 7.2 KPO4 buffer; (K&K) F6810 mM methionine; 10% L- glutamate AV071 40% sucrose 5% gelatin 0.02%Pluronic 25 mM, pH 7.2 KPO4 buffer; (K&K) F68 10 mM methionine AV047 5%sucrose; 2% HES 0.02% Pluronic 50 mM, pH 7.2 KPO4 buffer; W/HES 2%trehalose F68 10 mM methionine; 2 mM EDTA; 2% arginine AV069 10% sucrose2% HES 50 mM, pH 7.2 citrate buffer; 1 mM ZnCl2; 5% arginine AV047-P 5%sucrose; 0.02% Pluronic 50 mM, pH 7.2 KPO4 buffer; 2% trehalose F68 10mM methionine; 2 mM EDTA; 2% arginine AV047 5% sucrose; 0.02% Pluronic50 mM, pH 7.2 citrate buffer; citrate 2% trehalose F68 10 mM methionine;2 mM EDTA; 2% arginine

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims.

While the foregoing invention has been described in some detail forpurposes of clarity and understanding, it will be clear to one skilledin the art from a reading of this disclosure that various changes inform and detail can be made without departing from the true scope of theinvention. For example, the formulations, techniques and apparatusdescribed above can be used in various combinations. All publications,patents, patent applications, and/or other documents cited in thisapplication are incorporated by reference in their entirety for allpurposes to the same extent as if each individual publication, patent,patent application, and/or other document were individually indicated tobe incorporated by reference for all purposes.

1. A mixture of a suspension or solution with a high pressure gascomprising a pressure ranging from about 250 psi to a pressure less than90% of a critical pressure of the gas, the suspension or solutioncomprising a bioactive material, a polyol, a polymer, and a surfactant.2. The mixture of claim 1, wherein the bioactive material is selectedfrom the group consisting of proteins, peptides, nucleic acids,bacteria, cells, antibodies, enzymes, serums, vaccines, liposomes, andviruses.
 3. The mixture of claim 2, wherein the viruses are selectedfrom the list consisting of influenza virus, parainfluenza virus,respiratory syncytial virus, herpes simplex virus, cytomegalo virus,SARS virus, corona virus family members, human metapneumovirus, andEpstein-Bar virus.
 4. The mixture of claim 3, wherein the virus is alive virus present in the suspension or solution in a titer ranging fromabout 10¹ TCID₅₀ to about 10¹² TCID₅₀.
 5. The mixture of claim 1,wherein the bioactive material is present in an amount ranging fromabout 0.05 weight percent to about 1 weight percent of the suspension orsolution.
 6. The mixture of claim 1, wherein the polyol is selected fromthe group consisting of trehalose, sucrose, sorbose, melezitose,glycerol, fructose, mannose, maltose, lactose, arabinose, xylose,ribose, rhamnose, palactose, glucose, mannitol, xylitol, erythritol,threitol, sorbitol, and raffinose.
 7. The mixture of claim 6, whereinthe polyol is present in an amount ranging from about 1 weight percentto about 40 weight percent of the suspension or solution.
 8. The mixtureof claim 6, wherein the polyol is sucrose present in an amount of about10 weight percent of the suspension or solution.
 9. The mixture of claim6, further comprising one or more amino acids.
 10. The mixture of claim1, wherein the polymer is selected from the group consisting of starch,oxidized starch, carboxymethyl starch, hydroxyethyl starch (HES),hydrolyzed gelatin, polyvinyl pyrrolidone, unhydrolyzed gelatin,ovalbumin, collagen, chondroitin sulfate, a sialated polysaccharide,actin, myosin, microtubules, dynein, kinetin, and human serum albumin.11. The mixture of claim 10, wherein the polymer has a molecular weightranging from about 100 kDa to about 300 kDa.
 12. The mixture of claim10, wherein the polymer is present in a concentration ranging from about0.5 weight percent to about 10 weight percent of the suspension orsolution.
 13. The mixture of claim 12, wherein the polymer comprises HESpresent in a concentration of about 5 weight percent.
 14. The mixture ofclaim 1, wherein the surfactant is a nonionic surfactant selected fromthe group consisting of alkylphenyl alkoxylates, alcohol alkoxylates,fatty amine alkoxylates, polyoxyethylene glycerol fatty acid esters,castor oil alkoxylates, fatty acid alkoxylates, fatty acid amidealkoxylates, fatty acid polydiethanolamides, lanolin ethoxylates, fattyacid polyglycol esters, isotridecyl alcohol, fatty acid amides,methylcellulose, fatty acid esters, silicone oils, alkyl polyglycosides,glycerol fatty acid esters, polyethylene glycol, polypropylene glycol,polyethylene glycol/polypropylene glycol block copolymers, polyethyleneglycol alkyl ethers, polypropylene glycol alkyl ethers, polyethyleneglycol/polypropylene glycol ether block copolymers, polyethylene glycolsorbitan monolaurate, and polyoxyethylenesorbitan monooleate.
 15. Themixture of claim 1, wherein the surfactant is an ionic surfactantselected from the group consisting of alkylarylsulfonates,phenylsulfonates, alkyl sulfates, alkyl sulfonates, alkyl ethersulfates, alkyl aryl ether sulfates, alkyl polyglycol ether phosphates,polyaryl phenyl ether phosphates, alkylsulfosuccinates, olefinsulfonates, paraffin sulfonates, petroleum sulfonates, taurides,sarcosides, fatty acids, alkylnaphthalenesulfonic acids,naphthalenesulfonic acids, lignosulfonic acids, condensates ofsulfonated naphthalenes with formaldehyde, condensates of sulfonatednaphthalenes with formaldehyde and phenol, lignin-sulfite waste liquor,alkyl phosphates, quatemary ammonium compounds, amine oxides, andbetaines.
 16. The mixture of claim 1, wherein the surfactant is presentin an amount ranging from about 0.001 weight percent to about 5 weightpercent.
 17. The mixture of claim 16, wherein the surfactant is presentin an amount ranging from about 0.01 weight percent to about 1 weightpercent.
 18. The mixture of claim 1, wherein the suspension or solutionfurther comprises a buffer comprising a pH from about pH 3 to about pH8.
 19. The mixture of claim 18, wherein the buffer comprises a phosphatesalt, an amino acid, a carbonate salt, a borate salt, an acetate salt,histidine, glycine, or a citrate salt.
 20. The mixture of claim 18,wherein the buffer is present at a concentration ranging from about 2 mMto about 500 mM.
 21. The mixture of claim 1, wherein the suspension orsolution further comprises a carrier, excipient, or stabilizer.
 22. Themixture of claim 1, wherein the bioactive material comprises influenzavirus, the polyol comprises sucrose, the polymer comprises HES, and thesurfactant comprises a block copolymer of polyethylene and polypropyleneglycol.